A method of diagnosing cancer based on lipidomic analysis of a body fluid

ABSTRACT

A method of diagnosing cancer based on lipidomic analysis of a body fluid taken from the body of a patient is disclosed. The method includes the steps of spiking of the sample with a set of internal standards having at least one internal standard for each lipid class present in the sample, subsequently processing the sample by liquid-liquid lipidomic extraction or by solid phase lipidomic extraction, measurement of the processed sample by a mass spectrometry method, determining concentrations for at least 51, more preferably for all lipids present at a level above detection threshold of the mass spectrometry method, statistical evaluation of the determined concentrations of the lipids, the statistical evaluation determining the level of probability of the patient suffering from cancer, or optionally from a specific type of cancer.

FIELD OF ART

The present invention relates to a method of diagnosing cancer based onlipidomic analysis of a body fluid which is suitable for high throughputscreening with a high selectivity and specificity.

BACKGROUND ART

Cancer is one of the most serious human diseases, which results in hugenumber of deaths worldwide. There are numerous types of cancer,typically classified according to the primary organ, when the tumorcells start to grow. Even the cancer of one particular organ has severaldisease subtypes resulting in large complexity and heterogeneity of thisdisease, which requires a personalized treatment to increase the chancesfor a full or at least a partial recovery of individual patients. Earlydiagnosis of the cancer is the most critical issue investigated by manyresearch teams throughout the world, because the early cancer diagnosiscan significantly increase the chances for survival and recovery.Unfortunately, many cancer types have no or only minor symptoms at earlystages, so the common situation is that the patient is diagnosed toolate. The prognosis is much better when the disease is recognizedearlier, but the currently available diagnostic methods are complex,expensive, and laborious, which is unsuitable for routine populationscreening. Therefore, there is a need to continuously develop newmethods for screening and diagnosing cancer and for distinguishing amongvarious types of cancer, which would be fast, economical, with a limitedinvasiveness, suitable for routine high-throughput screening, with asufficient sensitivity and specificity, i.e., low percentage of falsepositive and false negative results. Various methods based on specificbiomarkers are investigated, using samples of body fluids, most commonlyblood. These tentative biomarkers are often proteins, glycoproteins,products of protein cleavage, and short RNA, such as miRNA. Thediagnosis using these biomarkers usually relies on immunochemicalmethods or PCR.

Breast cancer is besides skin cancer the most common cancer type forwomen and includes multiple sub-cancer types. Physical examination,mammography and fine-needle aspiration are common tools for thediagnosis of breast cancer. On the other hand, prostate cancer is themost common cancer type for men. Symptoms may be associated with urinarydysfunction. Another cancer type is kidney cancer. Their main subtypesare renal cell carcinoma, transitional cell cancer and Wilms tumor.Symptoms may involve blood in the urine, a lump in the abdomen andweight loss. The survival rate of all these cancer types stronglydepends on the cancer stage and on whether the cancer metastasized toother organs. In case of cancer suspicion, ultrasound, computertomography, or magnetic resonance imaging are employed. For cancerconfirmation needle biopsy is usually performed, whereby a cancer tissuesample is examined under the microscope.

Pancreatic cancer belongs among the most lethal tumors with the lowestsurvival rate of all cancers. It is expected to become the secondleading cause of cancer-related death in the US as well as Europe by theyear 2020. Pancreatic cancer is very hard to be diagnosed at earlystages. The pancreas is deep inside the body, so early tumors cannot beseen or felt by health care providers during routine physicalexaminations. People usually have no symptoms until the cancer hasalready spread to other organs. If people are at an increased risk ofpancreatic cancer due to genetic predispositions or show signs andsymptoms that can be associated with this disease, specific examinationsand tests can be done Imaging tests are most often performed in case ofserious suspicion of pancreatic cancer: computed tomography, magneticresonance imaging, abdominal or endoscopic ultrasound,cholangiopancreatography, somatostatin receptor scintigraphy, positronemission tomography, or angiography. Screening tests or examinations maybe used to look for a disease in people who have no symptoms, and whohave not had that disease before. However, the imaging tests used forpatients with an increased risk or a suspicion of pancreatic cancer arenot applicable for large-scale population screening, and moreoverimaging tests may have sensitivity limitations in the detection oftumors at an early stage. For a conclusive diagnosis of pancreaticcancer, percutaneous, endoscopic, or surgical biopsy can be done, butagain these invasive methods are not applicable for routine populationscreening.

So far, several types of blood tests have been considered to diagnosepancreatic cancer, such as carbohydrate antigen 19-9 (CA19-9),carcinoembryonic antigen (CEA), or Kirsten-ras (KRAS), but unfortunatelylow sensitivity and low specificity do not allow their use in theclinical practice for early diagnosis. No screening tests have so farbeen developed that would allow the detection of sufficiently earlystages to lower the risk of dying from pancreatic cancer. Reliabledetection of early stage pancreatic cancer is urgently needed, but atthis time, no major professional groups recommend any routine screeningfor pancreatic cancer in people at risk.

It would be desirable to develop diagnostic methods based on othermethodologies and other types of biomarkers. These screening methodshave to fulfil several requirements, such as available treatment forearly diagnosed disease with significantly better prognosis compared tolate diagnosis, sufficiently high sample throughput to be able toperform at least a partial population screening of individuals at higherrisk, the screening price has to be acceptable for the healthcare systemin relation to the benefits obtained by early diagnosis, etc. The bloodor urine collection is a common part of the preventative healthcare. Forthe population screening, a high throughput methodology based on bloodanalysis is indispensable. Once the blood analysis gives a positiveresult, further investigations such as imaging methods, i.e. magneticresonance spectroscopy, have to be done. However, even though such testis urgently needed, there are just limited studies so far for theanalysis of cancer from blood mainly based on the analysis of DNA, RNAor proteins. Previous studies showed that lipids play a crucial role incancer development, as demonstrated in the analysis of tumor tissues andcell lines [E. Cífková, M. Holčapek, M. Lísa, D. Vrána, J. Gatěk, B.Melichar, Anal. Bioanal. Chem. 407 (2015) 991-1002, E. Cífková, M. Lísa,R. Hrstka, D. Vrána, J. Gatěk, B. Melichar, M. Holčapek, Rapid Commun.Mass Spectrom. 31 (2017) 253-263], but not yet for collectable bodyfluids applicable for high-throughput screening. The first attemptstowards the lipidomic analysis of human body fluids have been presentedby Metanomics [US 2013/0140452, WO 2016/207391], but unfortunatelysensitivities and specificities were too low for real-life applications.The principal problem of body fluid analysis is that early stage tumormay have too small impact on the dysregulation of monitored metabolitesand lipids, which may result in the situation that the effect ofbiological variability is larger than the effect of the studied cancertype, and then no statistically relevant differences can be discoveredin body fluids.

The present invention aims at overcoming the problems and challenges ofthe current state of the art, and to provide a method of diagnosingcancer even in early stages, said method being usable for routinehigh-throughput population screening.

The notation of lipid compounds in this text is as follows: abbreviationof the class of the compounds, number of carbons: number of doublebonds. The abbreviation of the class may be preceded by informationabout isotopic labeling, if relevant. More details about the descriptionof lipid annotations and abbreviations used in this patent applicationis given in Table 1.

TABLE 1 Lipid nomenclature and abbreviations used in this application.Examples of lipid annotation for particular Lipid class/subclass LipidFatty acyl/alkyl Fatty acyl/alkyl Lipid category (abbreviation) specieslevel^(b) position level Glycerolipids Monoacylglycerols (MG) MG 18:0 MG18:0 MG Diacylglycerols (DG) DG 34:1 DG 18:1_16:0 DG Triacylglycerols(TG) TG57:3 TG TG Phosphatidyl- Diacyl^(c) PC 34:1 PC 16:0_18:1 PC16:0/18:1 cholines (PC) Alkyl-acyl or PC O-34:1 PC O-16:0_18:1 PCO-16:0/18:1 plasmalogen^(c) or PC P-PA 34:1 or PC P-PA 16:0_18:1 or PCP-PA 16:0/18:1 Glycero- Phosphatidic acids (PA) phospholipidsPhosphatidylethanolamines PE 34:1 PE 16:0_18:1 PE 16:0/18:1Phosphatidylglycerols (PG) PG 34:1 PG 16:0_18:1 PG 16:0/18:1Phosphatidylinositols (PI) PI 34:1 PI 16:0 18:1 PI 16:0/18:1Phosphatidylserines (PS) PS 34:1 PS 16:0 18:1 PS 16:0/18:1Lysophospholipids (prefix L) LPC 18:1 LPC 18:1 LPC 18:1/0:0 Ceramides(Cer) Cer 34:1 Cer d18:1/16:0 Sphingomyelins (SM) SM 34:1 SM d18:1/16:0Sphingolipids^(d) Hexosylceramides (HexCer) HexCer HexCer d18:1/16:0Dihexosylceramides Hex2Cer Hex2Cer d18:1/16:0 Sulfatides (Sul orSHexCer) SHexCer SHexCer d18:1/16:0 Sulfodihexosylceramides SHexCerSHexCer d18:1/16:0 Sterol Lipids Cholesteryl esters (CE) CE 16:0^(a)Information on the total number of carbon atoms and double bonds ofpresent fatty acyls, alkyls, or sphingoid backbone for particular lipidclass or subclass. ^(b)Particular information about present fatty acyls,alkyls, or sphingoid backbone for particular lipid class or subclass.^(c)Bond type subclasses of PC (applicable for other classes ofglycerophospholipids). ^(d)Annotation of lipid species level forsphingolipids is based on the common assumption of a sphingoid base withtwo hydroxyl groups and no hydroxylation of N-acyl. In case of thepresence of additional hydroxyl on the ceramide part of sphingolipidswithout any specification of its position, the OH in parentheses isplaced behind the CN:DB number.

SUMMARY OF THE INVENTION

The present invention provides a method using lipidomic analysis for thediagnosis of a plurality of human cancer types. The major advantage ofthe present invention is that the lipidomic profiling works well for allcancer stages including the most early stages, unlike a previouslypublished work based on the proteomic analysis combined with othermethodologies, where the sensitivity for stage 1 was only 40% [J. D.Cohen et al., Science 359 (2018) 926], which is truly not valuable forearly diagnosis. The basic and rather simple hypothesis explaining thesuccess of lipidomic analysis for cancer screening is that the tumorcells are so quickly divided that these cells require a large number ofbuilding blocks for the preparation of lipid bilayers of new tumorcells. This process is much faster than conventional cell division inthe healthy organism, therefore the lipidomic composition of tumor andnormal cell must be different as well, and these changes are alsoreflected in collectable body fluids.

The principal problem is that early stage tumors as well as differentcancer types may have too small impact on the dysregulation of monitoredmetabolites and lipids in body fluids. This may leads to the fact thatthe effect of biological variability is larger than the effect onstudied cancer types and then no statistically relevant differences canbe discovered in body fluids.

The present invention aims at overcoming the problems and challenges ofthe current state of the art, and to provide a method of diagnosingcancer from the analysis of biological fluids, which is also capable ofdistinguishing among multiple cancer types even in early stages, saidmethod being usable for routine high-throughput population screening.

The present invention thus provides a method of diagnosing cancer basedon lipidomic analysis of a body fluid taken from the body of a patient,comprising the steps of:

-   -   spiking of the sample with a set of internal standards        comprising at least one internal standard for each lipid class        present in the sample,    -   subsequently processing the sample by liquid-liquid lipidomic        extraction or by solid phase lipidomic extraction,    -   measurement of the processed sample by a mass spectrometry        method,    -   determining concentrations for at least 51, more preferably for        all lipids present at a level above detection threshold of the        mass spectrometry method, using the internal standards for the        corresponding lipid classes for the quantitation, or using        relative concentrations, or using ratios of concentrations        and/or signal intensities,    -   statistical evaluation of the determined concentrations of the        lipids, said statistical evaluation determining the level of        probability of the patient suffering from cancer, or optionally        from a specific type of cancer, based on the determined lipid        concentrations compared to a cancerous pattern, or optionally a        specific cancerous pattern, and a non-cancerous pattern, wherein        the cancerous pattern or the specific cancerous pattern and the        non-cancerous pattern are determined by statistical analysis of        the lipid concentrations for a group of known cancer samples and        non-cancer samples, and wherein the statistical evaluation is        done separately for males and females.

The method of the invention allows to distinguish between a healthyperson and a person suffering from a cancer, as well as to distinguishbetween various types of cancer. The cancerous pattern generallyindicates that the patient suffers from a cancer, while the specificcancerous pattern indicates that the patient suffers from a specifictype of cancer.

In one embodiment of the present invention, a method of diagnosingpancreaticcancer based on lipidomic analysis of a body fluid taken fromthe body of a patient is provided, comprising the steps of:

-   -   spiking of the sample with a set of internal standards        comprising at least one internal standard for each lipid class        present in the sample,    -   subsequently processing the sample by liquid-liquid lipidomic        extraction,    -   measurement of the processed sample by a mass spectrometry        method,    -   determining concentrations for at least 31 or for at least 51        lipids, preferably at least 60 or at least 120 lipids or at        least 230, more preferably for all lipids present at a level        above detection threshold of the mass spectrometry method, using        the internal standards for the corresponding lipid classes for        the quantitation, or using relative concentrations, or using        ratios of concentrations and/or signal intensities,    -   statistical evaluation of the determined concentrations of the        lipids, said statistical evaluation determining the level of        probability of the patient suffering from pancreatic cancer        based on the determined lipid concentrations compared to a        pancreatic cancerous pattern and a non-cancerous pattern,        wherein the cancerous pattern and the non-cancerous pattern are        determined by statistical analysis of the lipid concentrations        for a group of known pancreatic cancer samples and non-cancer        samples, and wherein the statistical evaluation is done        separately for males and females.

In this embodiment, the at least 31 lipids useful in method ofdiagnosing pancreatic cancer include:

Lipid HexCer d18:1/16:0; HexCer d18:1/15:1 (1OH) SM 34:1 HexCerd18:1/24:1; HexCer d18:1/23:2 (1OH) PE 34:1; PE P-35:0 PC 32:0; PCO-33:0 PE 36:4; PE P-37:3 PC 34:1; PC P-35:0 PE 34:2; PE P-35:1 PCP-34:0; PC 33:1 DG 36:2 Cer d18:1/24:1; Cer d18:1/23:2 (1OH) CE 16:0 SM41:1 SM 41:2 PC 34:4; PC P-35:3 HexCer d18:1/24:0; HexCer d18:1/23:1(1OH) PE P-36:3; PE 35:4 PE P-38:4; PE 37:5 PE P-38:5; PE 37:6 PEP-36:2; PE 35:3 SM 32:1 HexCer d18:1/22:0; HexCer d18:1/21:1 (1OH) PEP-38:3; PE 37:4 PC P-38:2; PC 37:3 PE P-40:5; PE 39:6 PE 36:0; PE P-38:6PE 38:0; PE P-40:6, PE 0-39:0 PE P-36:3; PE 35:4 PC P-34:1; PC 33:2 PCP-36:2; PC 35:3 DG 34:3

The term “body fluid” includes body fluids and components of bodyfluids. The body fluid for use in this invention is preferably selectedfrom plasma, serum, blood, urine, oncosomes, exosomes, extracellularvesicles. The isolation of exosomes and other extracellular vesicles(EV) from body fluids (onkosomes and microvesicles) or cell-culturemedia is known in the art and typically performed by differentialcentrifugation or ultracentrifugation, often combined with sucrosedensity gradients.

In preferred embodiments, the concentrations of at least 51, or at least120, or at least 230, or at least 400, or at least 406, or at least 440,or at least 450, or at least 500 lipids are determined.

In one preferred embodiment, the concentrations of at least 51 lipidsare determined when the body fluid is blood, plasma or serum.

A particularly preferred set of at least 51 lipids, the concentration ofwhich shall be determined in the methods of the present invention, isshown in Table 2 below. A more preferred set of lipids is shown in Table3 below. These lipids are particularly useful when the body fluid isblood, plasma or serum.

In another preferred embodiment, the concentrations of at least 75lipids are determined when the body fluid is urine. A particularlypreferred set of at least 75 lipids is shown in Table 4 below.

The patient is a mammal, preferably a human.

The specific types of cancer include kidney, prostate, breast, lungs,stomach, pancreatic, ovarian, head and neck, colon, liver, colorectal,esophagus, small intestine cancer, bladder, brain, cervical, thyroid,laryngeal, skin cancers, etc.

Preferably, the specific types of cancer include kidney, prostate,breast, lungs, pancreatic and stomach cancers.

The internal standards must be lipid-type compounds, structurally closeto the relevant lipid class. Typically, the internal standards arecompounds having the structure (in particular the polar head structure)typical for the relevant lipid class but containing fatty acyls withshorter chains than naturally occurring lipids (e.g. chains 12:0 or14:0) or fatty acyls with odd number of carbon atoms (e.g. chains 17:0,17:1 or 19:1) or isotopically labelled analogues (e.g. D7-Chol, D7-CE16:0).

The lipid classes are as follows:

Cholesterylesters Lysophosphatidylserines (CE) (LPS) CeramidesMonoacylglycerols (Cer) (MG) Diacylglycerols Phosphatidic acids (DG)(PA) Dihexosylceramides Phosphatidylcholines (Hex2Cer) (PC)Hexosylceramides Phosphatidylethanolamines (HexCer) (PE) CholesterolPhosphatidylglycerols (PG) Lysophosphatidic acids Phosphatidylserines(LPA) (PS) Lysophosphatidylcholines Sphingomyelins (LPC) (SM)Lysophosphatidylethanolamines Sulfohexosylceramides (LPE) (SulfoHexCer)Lysophosphatidylglycerols Triacylglycerols (LPG) (TG)

An example of a set of internal standards (IS) is the following list:

D7-CE 16:0 LPE 14:0 PG 14:0/14:0 Cer d18:1/12:0 LPG 14:0 PS 14:0/14:0 DG12:1/12:1 LPA 14:0 SM d18:1/12:0 Hex2Cer d18:1/12:0 MG 19:1 SulfoHexCerd18:1/12:0 HexCer d18:1/12:0 PA 14:0/14:0 TG 19:1/19:1/19:1 D7-Chol PC14:0/14:0 LPS 17:1 LPC 17:0 PE 14:0/14:0

The notation of lipid compounds in this text is as follows: abbreviationof the class of the compounds, number of carbons: number of doublebonds. The abbreviation of the class may be preceded by informationabout isotopic labeling, if relevant.

Due to the addition of the internal standard before sample processing,slight differences due to for instance pipetting errors can becompensated as the internal standard is affected in the same way as thetarget compounds. This ensures a better reliability of the quantitationof individual lipids.

Liquid-liquid lipidomic extractions are known in the art. Solvents maybe used, selected from chlorinated alkanes, dialkyl ethers, alcohols,and water mixtures, which forms bilayer systems containing organic andaqueous phases. Preferably, chloroform, methyl-tert-butyl ether,methanol, ethanol, propanol, and/or butanol are used. Most preferably,chloroform-methanol-water system is used, as described e.g. in E.Cífková, M. Holčapek, M. Lísa, D. Vrána, J. Gatěk, B. Melichar, Anal.Bioanal. Chem. 407 (2015) 991-1002; or in E. Cífková, M. Lísa, R.Hrstka, D. Vrána, J. Gatěk, B. Melichar, M. Holčapek, Rapid Commun. MassSpectrom. 31 (2017) 253-263. The organic phase of liquid-liquidextraction system containing lipids is collected and used for furtherprocessing. As the migration and solubility of the components of thesample are time-dependent, it is highly preferred that the extractionstep is always performed in the same way for all the samples measured inone batch, in particular that the extraction is performed over the sameperiod of time for each sample. The organic phase is typically processedby evaporation of the organic solvent and re-dissolving the residue inthe solvent for mass spectrometry measurement. Suitable solvents formass spectrometry measurements include chlorinated alkanes, alkanes andalcohols, preferably chloroform or dichloromethane, hexane and C1-C4alcohols, most preferably a mixture of chloroform and 2-propanol (e.g.,in volume ratio 1:1) or hexane:2-propanol:chloroform 7:1.5:1.5 orchloroform-methanol-2-propanol (1:2:4, v/v/v). Further components may beadded, e.g., ammonium acetate, acetic acid, etc. Alkanes, unless definedotherwise, mean C1-C6 alkanes, preferably C1-C4 alkanes.

It is preferred to remove proteins from the sample during or after theliquid-liquid extraction, as proteins may undesirably interfere with theanalysis of lipids.

The mass spectrometry method is selected from shotgun mass spectrometry,liquid chromatography-mass spectrometry (LC/MS), ultrahigh-performanceliquid chromatography-mass spectrometry (UHPLC/MS), supercritical fluidchromatography-mass spectrometry (SFC/MS), ultrahigh-performancesupercritical fluid chromatography-mass spectrometry (UHPSFC/MS), andmatrix-assisted laser desorption/ionization mass spectrometry(MALDI/MS).

The method of the present invention benefits from the use of a pooledsample. A pooled sample is a sample prepared by mixing identical volumesof several samples, wherein the mixed samples include samples fromcancer patients and healthy volunteers. The ratio of samples originatingfrom males to samples originating from females should be approximatelythe same as in the measured batch of samples. The term “approximatelythe same” refers to deviation from the ratio of male:female in themeasured batch at most by 20%, preferably at most by 10%, morepreferably at most by 5%. The pooled sample is preferably obtained bypooling 50-100 samples.

Concentrations of internal standards in the mixture were carefullyresearched in order to be applicable for all mass spectrometry basedmethods used in this invention. The concentration of the internalstandard should be preferably in the concentration range of naturallyoccurring lipids. However, as the concentration range variessignificantly within the lipid class, it is preferred to use theconcentration between 10 and 100% of the concentration of the mostabundant naturally occurring lipid species within the respective class,more preferably between 20 and 80%.

The pooled sample, processed in the same way as any other sample, i.e.,spiked with internal standard mixture and subjected to liquid-liquidextraction and any other sample processing steps, is used for the fullvalidation and quality control during the mass spectrometrymeasurements. The order of samples is randomized in sample measurementsequences to avoid measurements of non-cancerous and cancerous samplesin a certain portion of the measurement sequence.

The pooled sample may be used for observing intra-day accuracy andintra-day precision, as well as inter-day accuracy and inter-dayprecision.

Furthermore, it is advantageous to prepare test samples or pooledsamples spiked with varying concentrations of the internal standardmixture.

The limit of detection (LOD) of a lipid is preferably determined basedon signal to noise ratio, e.g., such as S/N=3.

The pooled sample spiked with varying concentrations of the internalstandard mixture is used for determining the lower limit of quantitation(LLOQ) and the upper limit of quantitation (ULOQ), said limitscorresponding to the first and the last points of linearity range of thecalibration curve. The calibration curve is the dependence of signal onthe concentration of a lipid. The linearity range of calibration curveis an interval from LLOQ to ULOQ, where the concentration of lipids canbe determined.

The concentrations for all lipids present at a level above LLOQ of themass spectrometry method are also commonly called “lipidomic profile”.

The quantitation (determining concentrations) for all lipids present ata level above LLOQ and below ULOQ of the mass spectrometry method, usingthe internal standards for the corresponding lipid classes for thequantitation, preferably comprises the following procedures andcorrections:

-   -   isotopic correction (deisotoping), i.e., correction of signal        intensity of a lipid having due to isotopic contribution M+2 of        another lipid with a molecular weight being 2 mass units lower        than said lipid (e.g., isotopic interference from the lipid with        one additional double bond) or due to isotopic contribution M+1        of another lipid with a molecular weight 1 mass unit lower than        said lipid (e.g., interferences between PC and SM subgroups).        The correction is done by subtracting calculated M+2 (or M+1)        relative intensity based on known intensity in measured spectra.    -   zero filling procedure in which the signals of lipid species        which are not detected for particular sample, are replaced by 50        to 100%, preferably by 60 to 100%, more preferably by 60 to 70%        or by 80 to 90%, or by 70 to 100% or by 67% or by 80%, of the        minimum concentration observed for said lipid species in all        samples.

The accuracy and reliability of quantitation is ensured or improved bythe following features:

-   -   use of at least one exogenous lipid class internal standard for        the quantitation of lipid species in each class,    -   coelution and coionization of lipid (sub)class internal        standards and analytes from the same lipid class using either        lipid class separation chromatography method coupled to mass        spectrometry (MS) or MS without any chromatographic separation.        This co-processing is ensured by the addition of the internal        standard before the sample is processed,    -   use of the pooled sample spiked with the internal standards as        quality control sample injected after a predetermined number of        samples,    -   measurement control by extracting the signal response of each        internal standard in each sample and evaluation of signal        response of those over the time period of the measurement        sequence daily,    -   zero filling procedure,    -   isotopic correction.

These features prevent one or more of: low reproducibility, lack ofrobustness, signal shifts over time, ion suppression/enhancement,carry-over effects, and response change of mass spectrometer due tocontamination.

These features thus help to control the quality of measurements byprevention of low reproducibility, lack of robustness, signal shiftsover time, ion suppression/enhancement, carry-over effects, and responsechange of mass spectrometer due to contamination.

When several hundreds of lipids are determined in hundreds of samples,it may happen that the expected signal is not detected for some lipidmolecules due to various reasons, and then the concentration should bereported as “below LLOQ” in accordance with established analyticalpractice. This well-established analytical approach does not work wellin case of lipidomic quantitation, and the MDA based on such approachwould provide poor resolution of healthy/disease groups, and may notprovide acceptable sensitivity and selectivity for the detection ofcancer. This approach is adapted to cases in which all missing signalscorrespond to the situation that the true concentration of the analyteis below LLOQ, for example the determination of low number of analyteswell separated by chromatography. However, the situation in lipidomicquantitation is very different, because quantify ca. 150-400 lipids persample are quantified and many of them (or all in case of shotgun) areco-eluting, which may result in the absence of signal due to otherreasons than the concentration below LLOQ, mainly due to ion suppressionor other reasons causing the signal drop, including the systemcontamination, mobile phase impurity, bleeding of chromatographiccolumn, etc. The complexity of lipidomic analysis is enormous, and it isa natural situation that some signals are not properly recorded andprocessed regardless of the best analytical practice and carefulanalytical work. Therefore, we have developed a new zero fillingapproach where the values which are missing for any reason are replacedby 75 to 85%, preferably by 80%, of the minimum concentration observedfor this lipid species in all samples. This approach significantlyimproves the quality of subsequent statistical analysis. Our preliminaryresults show that when the zero-filling approach is neglected, thequality of cancer detection is significantly worse or impossible.Afterwards the average and the standard deviation of the duplicates arecalculated.

The statistical evaluation is preferably performed by multivariate dataanalysis (MDA). The MDA methods may be non-supervised statisticalmethods such as principal component analysis (PCA) or supervisedstatistical methods such as orthogonal projections to latent structuresdiscriminant analysis (OPLS-DA).

The “cancerous pattern” means the pattern of lipid concentrationstypical for samples of a body fluid obtained from patients sufferingfrom a cancer. It is usually obtained by statistical analysis of thesamples obtained from patients suffering from various types of cancerversus samples obtained from healthy volunteers.

The “specific cancerous pattern” means the pattern of lipidconcentrations typical for samples of a body fluid obtained frompatients suffering from a specific type of cancer. It is usuallyobtained by statistical analysis of the samples obtained from patientssuffering from the specific type of cancer versus samples obtained fromhealthy volunteers, and/or versus samples obtained from patientssuffering from other specific types of cancer.

The “pancreatic cancerous pattern” means the pattern of lipidconcentrations typical for samples of a body fluid obtained frompatients suffering from pancreatic cancer. It is usually obtained bystatistical analysis of the samples obtained from patients sufferingfrom pancreatic cancer versus samples obtained from healthy volunteers.

The “non-cancerous pattern” means the pattern of lipid concentrationstypical for samples of a body fluid obtained from healthy volunteers,i.e., from subjects not suffering from cancer. The term “non-canceroussample” or “non-cancer sample” refers to a sample of a body fluidobtained from healthy volunteers, i.e., from subjects not suffering fromcancer.

Preferably, the statistical evaluation involves

-   -   data pre-processing such as centering, scaling, and/or        transformation. The centering correlates the changes relative to        the average of the data set. The scaling unifies the impact of        different concentration ranges on the model (Unit Variance (UV)        and Pareto scaling) so that low concentrations (maybe equally        important) are not masked by very abundant concentrations. The        logarithmic transformation assembles the data to the normal        distribution. A proper pre-processing has a significant        influence on the quality of results.    -   PCA analysis used for identification of outliers, measurement        failures as well as other influential factors. The PCA analysis        may also visualize differences between groups as well as cluster        the quality control (QC) samples in comparison to other samples.    -   discrimination analysis (OPLS-DA) for group separation of cancer        patients and healthy volunteers. The discrimination analysis is        performed for males and females separately.    -   assignment of the statistical parameters as well as the        evaluation of the prediction power.

The statistical methods are first used to build statistical models(cancerous patterns, specific cancerous patterns, and non-cancerouspatterns) based on lipidomic profiles of body fluids of healthyvolunteers and diagnosed cancer patients. These statistical models arethen used for visualization of differences of the cancerous andnon-cancerous pattern, as well as for the determination of the level ofprobability of the patient suffering from cancer or from a specificcancer (such as pancreatic cancer), or being healthy, based on thedetermined lipid concentrations resulting in a cancerous pattern, aspecific cancerous pattern or a non-cancerous pattern, respectively.

Preferably, in the step of evaluation using statistical data, theevaluated sample is first evaluated for the probability of the evaluatedpatient suffering from cancer or being healthy, and subsequently,provided that the evaluated patient is determined to have a probabilityof suffering from cancer being above a pre-determined level (e.g., thepre-determined level may be 50%, or 60%, or 70%, or 80%, or 90%), theevaluated sample is evaluated for determining the specific cancer.

The sensitivity and specificity describe the prediction power of themethod to correctly assign samples from healthy volunteers or cancerpatients groups. The sensitivity and specificity values should be over70%, preferably over 80% or over 85%, more preferably over 90% forsamples of known and unknown classification. In some embodiments, thesensitivity and specificity values achieved are over 95% for samples ofknown and unknown classification.

Based on the data obtained by the inventors, the sensitivity andselectivity of the method of the invention for pancreatic cancer, forexample for OPLS-DA models, is 100% for samples with knownclassification (292 subjects tested), and 95% for samples with unknownclassification (73 subjects tested). Such sensitivity and selectivity isbetter than selectivity and sensitivity achievable by any other knownmethod for pancreatic cancer diagnosis. Data obtained by the inventorsalso show that patients with stages 1-2 (early stages) can be identifiedin the screening using the method of the invention. This represents asignificant advantage of the present method. The capability to identifyearly stage cancers results in identification of patients at a stagewhen the cancer can be successfully treated.

The sample throughput of the methodology of the invention is at least4000 samples or preferably at least 10 000 samples per year and one massspectrometry apparatus at current level of automation of standard massspectrometry labs. Known methods of multiplexing and higher automationmay further increase the sample throughput.

Suitable sets of lipids to be determined for diagnosing the presence ofa cancer disease, or for diagnosing a specific type of cancer areusually obtained by an analysis of all detectable lipids for a group ofhealthy volunteers and cancer patients. The lipids which are present indifferent amounts in the healthy vs. cancer samples or healthy vs.specific cancer samples or first specific cancer vs. second specificcancer samples are preferred for inclusion into the sets of lipids to bedetermined.

TABLE 2 The following table shows a minimum list of lipid species theconcentrations of which are determined in the present invention(preferably in blood, plasma or serum samples). Optionally, theconcentrations of further lipids may be determined. Sphingolipids Cer34:1 SM 34:2 SM 40:1 SM 42:2 Sul 40:1 Sul 42:1 Cer 42:1 SM 36:1 SM 40:2SM 42:3 Sul 40:1(OH) Sul 42:1(2OH) Cer 40:1 SM 36:2 SM 41:1 SM 43:1 Sul40:2(OH) Sul 42:1(OH) Cer 42:2 SM 38:1 SM 41:2 Sul 34:0(OH) Sul 41:1 SM34:1 SM 39:1 SM 42:1 Sul 34:2(OH) Sul 41:1(OH) Glycerophospholipids LPC16:0 LPC 18:2 PC 34:1 PC 36:3 PC O-34:2/P-34:1 PC O-36:5/P-36:4 LPC 18:0PC 32:0 PC 34:2 PC 36:4 PC O-34:3/P-34:2 PE 34:1 LPC 18:1 PC 32:2 PC36:2 PC 38:4 PC O-36:3/P-36:2 Glycerolipids TG 49:1 TG 50:4 TG 50:5 TG51:4 TG 53:4 TG 55:5

Minimum list of lipid species listed in Table 2 is particularly suitablefor the differentiation of cancer and healthy samples. In general, theprediction power reflected in the sensitivity and specificity of a modelis the higher the more species are included to build the statisticalmodel. Therefore, it is advantageous to quantify as many lipids aspossible in the biological fluid. It is possible to reduce the variablesi.e. by including just statistically relevant species as determined bythe P-value and/or the VIP value (p-value<0.05 and VIP value>1). For thegeneration of the minimum list of lipid, only those species are includedwhich fulfil both criteria when analysing various cancer types. Acompromise for all three tested MS based methods was performed, thus thelist presented in Table 2 is generally applicable in the method of thepresent invention.

TABLE 3 The following table shows a preferred set of lipids theconcentrations of which are preferably determined in the presentinvention in blood, plasma or serum samples. Optionally, theconcentrations of further lipids may be determined. GlycerophospholipidsLPC 15:0 PC 36:2 PC O-36:5 PE 37:3 PE O-36:6 PI 34:2 LPC 16:0 PC 36:3 PCO-37:1 PE 37:5 PE O-36:7 PI 34:3 LPC 16:1 PC 36:4 PC O-37:2 PE 37:6 PEO-37:1 PI 35:0 LPC 18:0 PC 36:5 PC O-37:3 PE 38:0 PE O-37:2 PI 35:1 LPC18:1 PC 37:3 PC O-38:2 PE 38:1 PE O-37:3 PI 36:1 LPC 18:2 PC 37:4 PCO-38:3 PE 38:2 PE O-38:0 PI 36:2 LPC 20:3 PC 37:5 PC O-38:4 PE 38:4 PEO-38:1 PI 36:3 LPC 20:4 PC 37:6 PC O-38:5 PE 38:5 PE O-38:2 PI 36:4 LPC22:6 PC 38:2 PC O-38:6 PE 38:6 PE O-38:3 PI 38:1 LPC O-16:0 PC 38:3 PCO-39:2 PE 38:7 PE O-38:4 PI 38:2 LPE 16:0 PC 38:4 PC O-39:3 PE 39:0 PEO-38:5 PI 38:3 LPE 18:0 PC 38:5 PC O-40:5 PE 39:4 PE O-38:6 PI 38:4 LPE18:1 PC 38:6 PC O-40:6 PE 39:5 PE O-39:0 PI 38:5 LPE 18:2 PC 39:5 PCO-40:9 PE 39:6 PE O-39:1 PI 40:0 LPE 20:4 PC 39:6 PE 32:1 PE 40:4 PEO-39:2 PI 40:4 LPE 22:5 PC 40:4 PE 33:1 PE 40:5 PE O-40:0 PI 40:5 LPE22:6 PC 40:5 PE 33:2 PE 40:6 PE O-40:4 PI 40:6 PC 30:0 PC 40:6 PE 34:0PE 40:7 PE O-40:5 PS 34:0 PC 32:0 PC O-33:0 PE 34:1 PE 41:2 PE O-40:6 PS36:1 PC 32:1 PC O-33:1 PE 34:2 PE 42:9 PE O-40:7 PS 36:4 PC 32:2 PCO-33:2 PE 34:3 PE O-33:1 PE O-40:8 PS 36:5 PC 34:1 PC O-34:1 PE 35:1 PEO-34:1 PE O-40:9 PS 37:0 PC 34:2 PC O-34:2 PE 35:2 PE O-34:2 PE O-42:2PS 38:1 PC 34:3 PC O-34:3 PE 35:3 PE O-35:1 PG 32:1 PS 38:2 PC 34:4 PCO-35:1 PE 35:5 PE O-35:2 PG 36:0 PS 38:3 PC 35:1 PC O-35:2 PE 36:1 PEO-35:3 PG 36:4 PS 38:4 PC 35:2 PC O-35:3 PE 36:2 PE O-36:1 PI 32:0 PS38:6 PC 35:3 PC O-36:1 PE 36:3 PE O-36:2 PI 32:1 PS 38:7 PC 35:4 PCO-36:2 PE 36:4 PE O-36:3 PI 33:0 PS 42:1 PC 35:5 PC O-36:3 PE 36:5 PEO-36:4 PI 33:1 PC 36:1 PC O-36:4 PE 37:0 PE O-36:5 PI 34:1 SphingolipidsCer 33:2 (OH) Hex2Cer 33:2 (OH) SM 34:0 SM41:3 Sul 38:2 Sul 42:3 (OH)Cer 34:1 Hex2Cer 34:1 SM 34:1 SM 42:1 Sul 38:2 (OH) SulfoHex2Cer 34:1Cer 34:2 Hex2Cer 39:1 (OH) SM 34:2 SM 42:2 Sul 40:0 (2OH) SulfoHex2Cer40:1 Cer 34:3 Hex2Cer 39:2 (OH) SM 35:1 SM 42:3 Sul 40:0 (OH)SulfoHex2Cer 42:1 Cer 35:3 (OH) Hex2Cer 40:1 SM 36:0 SM 42:4 Sul 40:1SulfoHex2Cer 42:2 Cer 36:2 Hex2Cer 41:3 (OH) SM 36:1 SM 43:1 Sul 40:1(OH) GM3 34:1 Cer 36:3 Hex2Cer 42:2 SM 36:2 SM 43:2 Sul 40:2 GM3 34:2Cer 36:4 HexCer 33:2 (OH) SM 37:0 SM 43:3 Sul 40:2 (OH) GM3 36:1 Cer39:2 (OH) HexCer 34:1 SM 37:1 Sul 32:1 (OH) Sul 41:1 GM3 36:2 Cer 40:1HexCer 39:2 (OH) SM 38:0 Sul 34:0 (OH) Sul 41:1 (OH) GM3 38:1 Cer 40:2HexCer 40:1 SM 38:1 Sul 34:1 Sul 41:2 GM3 38:2 Cer 40:2 (OH) HexCer 41:2(OH) SM 38:2 Sul 34:1 (OH) Sul 41:2 (OH) GM3 40:2 Cer 41:1 HexCer 41:3(OH) SM 39:1 Sul 34:2 Sul 42:0 (2OH) GM3 40:1 Cer 41:2 (OH) HexCer 42:1SM 39:2 Sul 34:2 (OH) Sul 42:1 GM3 41:1 Cer 41:3 (OH) HexCer 42:2 SM40:0 Sul 35:1 Sul 42:1 (2OH) GM3 41:2 Cer 42:1 HexCer 42:2 (OH) SM 40:1Sul 36:1 Sul 42:1 (OH) GM3 42:3 Cer 42:2 HexCer 43:1 SM 40:2 Sul 36:1(OH) Sul 42:2 GM3 42:2 Cer 42:2 (OH) SM 32:1 SM 40:3 Sul 37:1 Sul 42:2(2OH) GM3 42:1 Cer 42:3 SM 32:1 SM 41:1 Sul 38:1 Sul 42:2 (OH) GM3 42:2(OH) Cer 43:1 SM 33:1 SM 41:2 Sul 38:1 (OH) Sul 42:3 GM3 42:1 (OH)Glycerolipids MG 16:0 DG 36:1 TG 47:2 TG 50:5 TG 53:4 TG 56:4 MG 16:1 DG36:2 TG 48:0 TG 51:1 TG 53:5 TG 56:5 MG 18:0 DG 36:3 TG 48:1 TG 51:2 TG54:1 TG 56:6 MG 18:1 DG 36:4 TG 48:2 TG 51:3 TG 54:2 TG 56:7 MG 18:2 TG42:2 TG 48:3 TG 51:4 TG 54:3 TG 56:8 MG 19:0 TG 44:0 TG 48:4 TG 51:5 TG54:4 TG 57:1 DG 30:0 TG 44:1 TG 49:0 TG 51:6 TG 54:5 TG 58:0 DG 32:0 TG44:2 TG 49:1 TG 52:1 TG 54:6 TG 58:2 DG 32:1 TG 45:0 TG 49:2 TG 52:2 TG54:7 TG 58:3 DG 32:2 TG 45:1 TG 49:3 TG 52:3 TG 54:8 TG 58:4 DG 34:0 TG46:0 TG 50:0 TG 52:4 TG 55:4 TG 58:5 DG 34:1 TG 46:1 TG 50:1 TG 52:5 TG55:5 TG 58:6 DG 34:2 TG 46:2 TG 50:2 TG 52:6 TG 55:6 TG 58:7 DG 34:3 TG47:0 TG 50:3 TG 53:2 TG 56:1 TG 58:8 DG 36:0 TG 47:1 TG 50:4 TG 53:3 TG56:2 TG 58:8 Sterol lipids CE 14:0 CE 16:1 CE 18:2 CE 20:4 CE 22:6cholesterol sulfate CE 16:0 CE 18:1 CE 18:3 CE 20:5 cholesterolcholesterol sulfate (OH)

TABLE 4 The following table shows a preferred set of lipids theconcentrations of which are preferably determined in the presentinvention for urine samples. Optionally, the concentrations of furtherlipids may be determined. Sterol conjugates Sulfoglycosphinglolipidshydroxypregnenolone sulfate Sul 34:1 Sul 42:1 (2OH) lithocholic acidglycine conjugate Sul 34:1 (OH) Sul 42:0 (2OH) cortisol sulfate Sul 36:1Sul 43:1 (2OH) glycochenodeoxycholic acid Sul 36:1 (OH) Sul 43:0 (2OH)lithocholic acid sulfate Sul 38:1 SulfoHex₂Cer 34:1 testosteroneglucuronide Sul 38:1 (OH) SulfoHex₂Cer 34:1 (OH) glycocholic acid Sul40:2 SulfoHex₂Cer 36:1 androsterone glucuronide/dihydrotestosterone Sul40:1 SulfoHex₂Cer 38:1 glucuronide Sul 41:2 SulfoHex₂Cer 38:1 (OH)cholesterol sulfate Sul 40:2 (OH) SulfoHex₂Cer 40:2glycochenodeoxycholic acid sulfate Sul 41:1 SulfoHex₂Cer 40:1hydroxyandrostenedione glucuronide Sul 40:1 (OH) SulfoHex₂Cer 41:111-oxo-androsterone glucuronide Sul 40:0 (OH) SulfoHex₂Cer 40:1 (OH)hydroxyandrosterone glucuronide Sul 42:3 SulfoHex₂Cer 42:3taurolithocholic acid Sul 42:2 SulfoHex₂Cer 42:2 sulfocholic acid Sul41:2 (OH) SulfoHex₂Cer 42:1 pregnanediol glucuronide Sul 42:1SulfoHex₂Cer 41:1 (OH) taurodeoxycholic acid Sul 41:1 (OH) SulfoHex₂Cer40:0 (2OH) sulfoglycolithocholic acid Sul 41:0 (OH) SulfoHex₂Cer 42:2(OH) taurocholic acid Sul 40:0 (2OH) SulfoHex₂Cer 42:1 (OH)glycochenodeoxycholic acid sulfate Sul 42:3 (OH) SulfoHex₂Cer 41:0 (2OH)aldosterone glucuronide Sul 42:2 (OH) SulfoHex₂Cer 42:0 (2OH)tetrahydroaldosterone glucuronide Sul 42:1 (OH) psychosine sulfate Sul42:0 (OH) deoxycholic acid glucuronide Sul 41:0 (2OH) cholic acidglucuronide Sul 43:2 (OH) glycochenodeoxycholic acid glucuronide Sul43:1 (OH)

Preferred sets of lipids for individual types of cancers are shown inthe following tables.

TABLE 5 Most up- and down-regulated species from S-plot of OPLS-DA modelwhen comparing non-cancerous samples with cancerous samples for bothgenders and males (M) and various cancer types (N = non- tumor, T =tumor, data about sensitivity and specificity relate to a set of personswhose samples were measured employing UHPSFC/MS). N-T N-T N-T N-T N-Tall cancers all breast kidney prostate S-plot both genders cancers Mcancer M cancer M cancer M Up-regulated DG 34:0 DG 34:0 DG 34:0 DG 34:0TG 49:0 species in MG 18:0 DG 36:0 DG 36:0 DG 36:0 TG 42:2 cancer TG49:0 TG 49:0 MG 16:0 MG 18:0 DG 34:0 MG 16:0 MG 18:0 MG 18:0 MG 16:0 DG36:0 DG 36:0 TG 42:2 TG 50:0 TG 42:2 TG 44:0 Down-regulated TG 42:2 MG16:0 TG 48:0 TG 49:0 TG 56:1 species in TG 44:0 TG 44:0 TG 51:1 Cer 42:3TG 45:0 cancer SM 42:1 SM 42:1 SM 42:1 SM 42:1 SM 42:1 SM 41:1 SM 41:1LPC 18:2 SM 41:1 SM 41:1 SM 41:2 SM 40:1 LPC 18:0 TG 58:6 SM 40:1 SM40:1 LPC 18:2 LPC 18:1 LPC 18:2 LPC 18:2 SM 38:1 SM 38:1 LPC 16:0 SM40:1 LPC 18:0 SM 40:2 SM 41:2 SM 41:1 SM 38:1 SM 41:2 Cer 42:l SM 40:2SM 40:1 SM 41:2 SM 40:2 LPC 18:2 LPC 18:0 PC 32:2 Cer 42:1 SM 38:1 SM34:1 Cer 42:l SM 38:1 SM 40:2 LPC 16:0 LPC 18:0 SM 34:1 PC O-36:4 TG58:4 SM 42:2 No.: N/T 170/282 75/157 75/9 75/81 75/67 Sensitivity [%]89.4 93.0 100 90.1 86.6 Specificity [%] 80.6 81.3 100 90.7 94.7

TABLE 6 Most up- and down-regulated species from S-plot of OPLS-DA modelwhen comparing non-cancerous samples with cancerous samples for females(F) and various cancer types (N = non-tumor, T = tumor, data aboutsensitivity and specificity relate to a set of persons whose sampleswere measured employing UHPSFC/MS). N-T N-T N-T all breast kidney S-plotcancers F cancer F cancer F Up-regulated MG 18:0 MG 18:0 MG 18:0 speciesin MG 16:0 MG 16:0 MG 16:0 cancer DG 34:0 TG 49:0 DG 34:0 TG 49:0 DG34:0 DG 36:0 TG 42:2 TG 42:2 — DG 36:0 TG 44:0 — — TG 58:2 — SM 42:1 SM42:1 SM 42:1 SM 41:1 PC 36:1 SM 41:1 SM 41:2 SM 41:1 SM 41:2Down-regulated Cer 42:1 PC O-34:3 TG 56:1 species in SM 40:1 SM 41:2 PC32:2 cancer PC O-34:3 PC O-38:6 SM 40:1 SM 38:1 PC O-36:3 Cer 42:1 PC36:1 SM 40:1 TG 47:1 TG 55:5 Cer 42:1 TG 49:1 SM 40:2 SM 38:1 TG 51:4No.: N/T 95/125 95/94 95/31 Sensitivity [%] 88.0 87.2 74.2 Specificity[%] 87.4 93.7 100

TABLE 7 Most up- and down-regulated species from S-plot of OPLS-DA modelwhen comparing cancerous samples with each other (N = non-tumor, T =tumor, data about sensitivity and specificity relate to a set of personswhose samples were measured employing UHPSFC/MS). T-T T-T T-T 1/kidneyvs. 1/breast vs. 1/breast vs. S-plot 2/prostate M 2/kidney M 2/kidney FUp-regulated TG 56:1 SM 42:1 PC 36:1 species in TG 50:0 LPC 18:2 TG 58:6cancer 2 TG 44:1 PC 32:2 PC O-38:6 TG 44:0 LPC 18:1 PC O-38:5 TG 46:0LPC 18:0 Cer 42:3 TG 44:2 PC O-38:6 PC O-36:5 TG 46:1 LPC 16:0 Cer 40:1TG 45:0 PC O-36:3 PC 32:0 TG 42:2 PC O-34:3 DG 36:2 TG 47:0 PC 36:5 LPC18:1 Up-regulated MG 18:0 TG 50:0 TG 49:0 species in MG 16:0 TG 55:5 TG56:1 cancer 1 DG 36:0 TG 51:1 TG 44:1 DG 34:0 TG 58:6 TG 45:0 DG 36:3 TG48:0 TG 45:1 DG 34:2 TG 53:2 TG 44:2 DG 32:0 TG 52:1 TG 46:2 DG 32:1 TG56:1 TG 44:0 DG 36:2 TG 49:0 TG 47:1 DG 34:1 TG 54:2 TG 46:1 No.: T/T81/67 9/81 94/31 Sensitivity [%] 76.1 100 74.2 Specificity [%] 80.3 22.296.8

TABLE 8 Most up- and down-regulated species from S-plot of OPLS-DA modelwhen comparing non-cancerous samples with cancerous samples for bothgenders and males (M) and various cancer types (N = non-tumor, T =tumor, data about sensitivity and specificity relate to a set of personswhose samples were measured employing MALDI-MS). N-T N-T N-T N-T N-T allcancers all breast kidney prostate S-plot both genders cancers M cancerM cancer M cancer M Up-regulated — — — SulfoHex2Cer 42:2 —Down-regulated Sul 34:0 (OH) SM 43:1 Sul 40:1 SM 43:1 Sul 34:0 (OH)species in SM 43:1 SM 39:1 Sul 41:1 (OH) Sul 41:1 (OH) Sul 40:1 cancerSM 39:1 Sul 41:1 (OH) Sul 34:0 (OH) SM 39:1 SM 43:1 Sul 41:1 (OH) Sul40:1 Sul 42:1 Sul 40:1 (OH) Sul 42:1 Sul 40:1 Sul 34:0 (OH) Sul 40:1(OH) Sul 34:0 (OH) SM 39:1 SM 41:1 Sul 40:1 (OH) Sul 42:1 (OH) Sul 40:1SM 40:3 Sul 41:1 SM 41:1 Sul 40:2 (OH) SM 41:1 Sul 34:2 (OH) Sul 34:2(OH) Sul 34:2 (OH) Sul 34:2 (OH) Sul 42:1 (OH) Sul 40:2 (OH) Sul 42:1Sul 40:2 (OH) Sul 41:1 SM 32:1 Sul 41:1 (OH) SM 33:1 SM 32:1 Sul 42:3(OH) Sul 34:2 (OH) Sul 40:1 (OH) No.: N/T 170/282 75/157 75/9 75/8075/67 Sensitivity [%] 89.7 91.1 55.6 90.0 85.1 Specificity [%] 82.9 81.3100 93.3 96.0

TABLE 9 Most up- and down-regulated species from S-plot of OPLS-DA modelwhen comparing non-cancerous samples with cancerous samples for females(F) and various cancer types (N = non-tumor, T = tumor, data aboutsensitivity and specificity relate to a set of persons whose sampleswere measured employing MALDI-MS). N-T N-T N-T all breast kidney S-plotcancers F cancer F cancer F Up-regulated — — SulfoHex2Cer 42:2Down-regulated Sul 34:0 (OH) Sul 34:0 (OH) Sul 41:1 (OH) species in Sul41:1 (OH) SM 39:1 Sul 34:0 (OH) cancer SM 43:1 Sul 41:1 (OH) SM 39:1 SM39:1 SM 43:1 SM 43:1 Sul 41:1 SM 33:1 Sul 40:1 (OH) Sul 40:1 Sul 42:1Sul 42:1 (OH) Sul 42:1 Sul 40:1 Sul 42:1 (2OH) SM 33:1 Sul 41:1 Sul 41:1Sul 42:1 (2OH) SM 43:2 SM 41:1 Sul 34:2 (OH) SM 32:1 Sul 40:1 No.: N/T95/125 95/94 95/31 Sensitivity [%] 88.8 86.2 83.9 Specificity [%] 91.695.8 98.9

TABLE 10 Most up- and down-regulated species from S-plot of OPLS-DAmodel when comparing non- cancerous samples with cancerous samples forboth genders and males (M) and various cancer types (N = non-tumor, T =tumor, data about sensitivity and specificity relate to a set of personswhere result; from MALDI and UHPSFC are combined). N-T N-T N-T N-T N-Tall cancers all breast kidney prostate S-plot both genders cancers Mcancer M cancer M cancer M Up-regulated DG 34:0 DG 34:0 DG 34:0 DG 34:0TG 49:0 species in MG 18:0 DG 36:0 DG 36:0 DG 36:0 TG 42:2 cancer MG16:0 MG 18:0 MG 16:0 MG 18:0 TG 44:0 DG 36:0 MG 16:0 MG 18:0 MG 16:0 TG56:1 TG 49:0 TG 42:2 TG 50:0 SulfoHex2Cer 42:2 DG 36:0 TG 42:2 TG 49:0TG 48:0 TG 49:0 DG 34:0 TG 44:0 SulfoHex2Cer 42:2 TG 51:1 TG 42:2 TG45:0 — TG 44:0 TG 55:5 PI 38:6 — Down-regulated SM 42:1 SM 42:1 SM 42:1SM 42:1 SM 42:1 species in SM 41:1 SM 41:1 LPC 18:2 TG 58:6 SM 41:1cancer Sul 34:0 (OH) SM 40:1 LPC 18:0 SM 41:1 SM 40:1 SM 41:2 Sul 41:1(OH) Sul 40:1 Sul 41:1 (OH) LPC 18:0 SM 40:1 SM 43:1 LPC 18:1 SM 40:1LPC 18:2 SM 43:1 LPC 18:2 LPC 16:0 SM 43:1 SM 38:1 SM 38:1 Sul 40:1 Sul42:1 SM 38:1 SM 41:2 SM 40:2 SM 38:1 Sul 34:0 (OH) Sul 40:1 (OH) SM 40:2SM 39:1 Sul 34:0 (OH) Sul 41:1 (OH) SM 39:1 Sul 34:0 (OH) Sul 41:1 (OH)Sul 40:1 (OH) Sul 40:1 (OH) SM 41:2 Sul 40:1 No.: N/T 170/282 75/15775/9 75/81 75/67 Sensitivity [%] 92.6 94.3 100 93.8 79.1 Specificity [%]87.7 77.3 100 93.3 97.3

TABLE 11 Most up- and down-regulated species from S-plot of OPLS-DAmodel when comparing non-cancerous samples with cancerous samples forfemales (F) and various cancer types (N = non-tumor, T = tumor, dataabout sensitivity and specificity relate to a set of persons whereresults from MALDI and UHPSFC are combined). N-T N-T N-T all breastkidney S-plot cancers F cancer F cancer F Up-regulated MG 18:0 MG 18:0MG 18:0 species in MG 16:0 MG 16:0 MG 16:0 cancer DG 34:0 TG 49:0 DG34:0 TG 49:0 DG 34:0 DG 36:0 TG 42:2 TG 42:2 Cer 42:3 TG 44:0 TG 44:0SulfoHex2Cer 42:2 DG 36:0 TG 58:2 — — TG 58:3 — Down-regulated SM 42:1SM 42:1 SM 42:1 species in SM 41:1 SM 41:1 SM 41:1 cancer SM 41:2 SM41:2 Sul 41:1 (OH) SM 43:1 PC O-34:3 SM 41:2 TG 55:5 PC 36:1 SM 39:1 SM40:1 SM 40:1 Sul 42:1 (OH) Cer 42:1 Sul 42:1 Sul 40:1 (OH) Sul 41:1 (OH)Sul 34:0 (OH) TG 51:4 PC O-34:3 PC O-36:3 TG 49:2 SM 39:1 Sul 41:1 (OH)PC 32:2 No.: N/T 95/125 95/94 95/31 Sensitivity [%] 89.6 91.5 83.9Specificity [%] 90.5 94.7 100

TABLE 12 Most up- and down-regulated species from S-plot of OPLS-DAmodel when comparing cancerous samples (cancer types) with each other (N= non-tumor, T = tumor, data about sensitivity and specificity relate toa set of persons where results from MALDI and UHPSFC/MS are combined).T-T T-T T-T 1/kidney vs. 1/breast vs. 1/breast vs. S-plot 2/prostate M2/kidney M 2/kidney F Up-regulated TG 56:1 SM 42:1 PC 36:1 species in TG50:0 LPC 18:2 PC O-38:6 cancer 2 TG 44:0 PC 32:2 PC O-38:5 TG 45:0 Sul42:1 PC O-36:5 TG 49:0 Sul 40:1 Cer 42:3 TG 46:0 LPC 18:1 SM 36:0 TG44:1 LPC 18:0 TG 58:6 TG 47:0 Sul 40:2 PC 32:0 TG 46:1 Sul 42:1 (OH) Cer40:1 TG 44:2 Sul 42:2 DG 36:0 Up-regulated MG 18:0 TG 55:5 TG 49:0species in MG 16:0 TG 58:6 TG 56:1 cancer 1 DG 36:0 TG 53:2 TG 48:4 DG34:0 TG 56:5 TG 44:1 DG 36:3 TG 51:1 TG 50:5 SulfoHex2Cer 42:2 TG 53:3TG 52:6 DG 34:2 DG 34:0 TG 48:3 Sul 42:3 TG 55:4 TG 46:2 LPC 16:0 TG54:2 TG 50:4 DG 34:1 TG 56:6 TG 44:2 No.: T/T 81/67 9/81 94/31Sensitivity [%] 91.0 100 77.4 Specificity [%] 82.7 22.2 97.9

TABLE 13 Most up- and down-regulated species from S-plot of OPLS-DAmodel when comparing non-cancerous plasma samples with kidney cancerousplasma samples for males (M) and females (F) (N = non-tumor, T = tumor,data about sensitivity and specificity relate to a set of persons whosesamples were measured employing shotgun MS). N-T N-T kidney kidneyS-plot F M Up-regulated PE 42:9 LPC 16:0 species in SM 42:2 SM 42:2cancer PC 38:4 Hex2Cer 34:1 MG 19:0 PS 42:1 LPC O-16:0 PS 36:5 TG 56:5HexCer 42:2 PS 32:2 CE 16:0 SM 36:0 PC 34:1 CE 16:0 PS 36:4 LPC 18:0 MG18:0 Down-regulated CE 14:0 LPC 18:2 species in PC 32:2 PE P-36:4 cancerSM 33:1 SM 39:1 SM 32:1 PC 34:4 PC 34:4 SM 41:2 SM 39:1 PC O-36:5 SM41:1 PS 38:7 PC 34:3 SM 41:1 HexCer 42:1 TG 56:6 LPC 18:2 SM 40:2 No.:T/N 28/31 71/30 Sensitivity [%] 100 95.8 Specificity [%] 93.6 76.7

TABLE 14 Most up- and down-regulated species from S-plot of OPLS-DAmodel when comparing non-cancerous (N) urine samples with kidney andprostate cancerous (T) urine samples for both genders, males (M) andfemales (F) (N = non-tumor, T = tumor, data about sensitivity andspecificity relate to a set of persons whose samples were measuredemploying MALDI-MS (absolute quantitation). N-T both cancers N-T N-T(kidney, prostate) prostate kidney S-plot M cancer M cancer MUp-regulated Sul 42:3 Sul 42:3 SulfoHex2Cer 40:2 species in SulfoHex2Cer40:2 SulfoHex2Cer 38:1 (OH) Sul 42:3 cancer SulfoHex2Cer 38:1 (OH)cortisol sulfate SulfoHex2Cer 38:1 sulfocholic acid SulfoHex2Cer 34:1(OH) sulfocholic acid Sul 43:1 (2OH) Sul 43:1 (2OH) SulfoHex2Cer 42:2SulfoHex2Cer 34:1 (OH) SulfoHex2Cer 40:2 SulfoHex2Cer 38:1 (OH) Sul 42:3(OH) SulfoHex2Cer 41:1 (OH) SulfoHex2Cer 34:1 SulfoHex2Cer 41:1 (OH)sulfocholic acid SulfoHex2Cer 42:1 cortisol sulfate Sul 42:3 (OH)SulfoHex2Cer 34:1 (OH) SulfoHex2Cer 38:1 Sul 41:2 Sul 43:1 (2OH)Down-regulated Sul 38:1 (OH) hydroxypregnenolone sulfate Sul 40:0 (2OH)species in Sul 34:1 (OH) Sul 38:1 (OH) Sul 41:0 (2OH) cancerSulfoHex2Cer 40:1 (OH) Sul 40:2 Sul 41:0 (OH) Sul 40:0 (2OH)SulfoHex2Cer 40:1 (OH) Sul 38:1 (OH) Sul 40:1 (OH) Sul 38:1 Sul 40:0(OH) SulfoHex2Cer 40:0 (2OH) SulfoHex2Cer 40:0 (2OH) Sul 40:1 (OH) Sul41:0 (OH) Sul 34:1 (OH) Sul 42:0 (2OH) Sul 41:1 Sul 42:1 (2OH) Sul 42:1(2OH) Sul 41:0 (2OH) Sul 40:1 (OH) Sul 41:1 (OH) taurolithocholic acidSul 41:1 No.: N/T 34/137 34/67 34/72 Sensitivity [%] 93.5 91.0 91.7Specificity [%] 58.8 76.5 76.5 N-T N-T kidney cancer kidney S-plot bothgenders cancer F Up-regulated SulfoHex2Cer 42:2 Sul 42:3 species in Sul42:3 SulfoHex2Cer 40:2 cancer SulfoHex2Cer 40:2 SulfoHex2Cer 42:2SulfoHex2Cer 38:1 SulfoHex2Cer 38:1 (OH) SulfoHex2Cer 42:1 SulfoHex2Cer42:1 cortisol sulfate Sul 43:1 (2OH) SulfoHex2Cer 40:1 Sul 42:3 (OH)SulfoHex2Cer 34:1 SulfoHex2Cer 40:1 SulfoHex2Cer 38:1 (OH) Sul 42:2sulfocholic acid SulfoHex2Cer 41:1 (OH) Down-regulated taurolithocholicacid taurolithocholic acid species in Sul 40:0 (2OH) taurocholic acidcancer Sul 41:0 (2OH) taurodeoxycholic acid Sul 38:1 (OH)sulfoglycolithocholic acid cholesterol sulfate Sul 40:0 (2OH) Sul 41:0(OH) Sul 34:1 (OH) Sul 40:0 (OH) Sul 41:0 (2OH) SulfoHex2Cer 40:0 (2OH)Sul 38:1 (OH) Sul 42:0 (2OH) Sul 40:0 (OH) Sul 40:1 (OH) Sul 42:1 (2OH)No.: N/T 70/101 36/29 Sensitivity [%] 87.1 86.2 Specificity [%] 87.186.1

For diagnosing pancreatic cancer, the lipidomic profile comprising atleast 51, preferably at least 120, lipids is used in the present method.In one preferred embodiment, the lipids to be detected in order to builda very reliable statistical model are preferably at least the sets shownin Table 15, or the sets listed in the Tables 28, 29, 30 or 31.

TABLE 15 Set 2 (particularly suitable for Set 1 (particularly suitablefor shotgun MS) UHPSFC/MS) CE 14:0 PE 32:0; PE O-33:0 CE 16:1 CE 16:0 PE32:1; PE P-33:0 CE 16:0 CE 16:1 PE 34:0; PE P-36:6 CE 18:3 CE 18:1 PE34:1; PE P-35:0 CE 18:2 CE 18:2 PE 34:2; PE P-35:1 CE 18:1 CE 18:3 PE34:3; PE P-35:2 CE 20:5 CE 20:4 PE 36:0; PE P-38:6 CE 20:4 CE 20:5 PE36:1; PE P-37:0 CE 20:3 CE 22:4 PE 36:2; PE P-37:1 CE 20:2 CE 22:5 PE36:3; PE P-37:2 CE 22:6 CE 22:6 PE 36:4; PE P-37:3 Cer d42:2 Cerd18:0/16:1; Cer d18:0/15:2 (1OH) PE 36:5; PE P-37:4 Cer d42:1 Cerd18:0/16:2 PE 38:0; PE P-40:6, PE O-39:0 DG 34:2 Cer d18:0/18:2 PE 38:1;PE P-40:7; PE P-39:0 DG 34:1 Cer d18:0/24:0; Cer d18:0/23:1 (1OH) PE38:2; PE P-40:8; PE P-39:1 DG 36:3 Cer d18:0/24:1; Cer d18:0/23:2 (1OH)PE 38:3; PE P-40:9; PE P-39:2 DG 36:2 Cer d18:1/16:0; Cer d18:1/15:1(1OH) PE 38:4; PE P-40:10; PE P-29:3 DG 36:0 Cer d18:1/16:1; Cerd18:1/15:2 (1OH) PE 38:5; PE P-39:4 DG 37:4 Cer d18:1/16:2 PE 38:6; PEP-39:5 Coenzyme Q10 Cer d18:1/18:0; Cer d18:1/17:1 (1OH) PE 38:7; PEO-38:0; PE 37:0 MG 16:0 Cer d18:1/18:2 PE 40:1; PE P-42:7; PE P-41:0 MG18:1 Cer d18:1/18:3 PE 40:2; PE P-42:8; PE P-41:1 MG 18:0 Cerd18:1/20:0; Cer d18:1/19:1 (1OH) PE 40:3; PE P-42:9; PE P-41:2 PCO-32:1/P-32:0 Cer d18:1/22:0; Cer d18:1/21:1 (1OH) PE 40:4; PE P-42:10;PE P-41:3 PC 32:2 Cer d18:1/23:0; Cer d18:1/22:1 (1OH) PE 40:5; PEP-41:4 PC 32:1 Cer d18:1/24:0; Cer d18:1/23:1 (1OH) PE 40:6; PE P-41:5PC 32:0 Cer d18:1/24:1; Cer d18:1/23:2 (1OH) PE 40:7; PE O-40:0; PE 39:0PC O-34:3/P-34:2 Cer d18:1/25:0; Cer d18:1/24:1 (1OH) PE 40:8; PEP-40:0; PE 39:1 PC 34:3 DG 30:0 PE 42:9; PE P-42:1; PE 41:2 PC 34:2 DG31:0 PEP-34:0; PE 33:1 PC 34:1 DG 32:0 PE P-34:1; PE 33:2 PCO-36:5/P-36:4 DG 32:1 PE P-36:0; PE 35:1 PC O-36:4/P-36:3 DG 32:2 PEP-36:1; PE 35:2 PC O-36:3/P-36:2 DG 34:0 PE P-36:2; PE 35:3 PC 35:2 DG34:1 PE P-36:3; PE 35:4 PC 35:1 DG 34:2 PE P-36:4; PE 35:5 PC 36:5 DG34:3 PE P-38:2; PE 37:3 PC 36:4 DG 36:0 PE P-38:3; PE 37:4 PC 36:3 DG36:1 PE P-38:4; PE 37:5 PC 36:2 DG 36:2 PE P-38:5; PE 37:6 PC 36:1 DG36:3 PE P-40:4; PE 39:5 PC O-38:6/P-38:5 DG 36:4 PE P-40:5; PE 39:6 PCO-38:5/P-38:4 DG 38:2 PG 32:0; PG O-33:0 PC 37:3 DG 38:3 PG 32:1; PGP-33:0 PC 38:7 DG 38:4 PG 34:0; PG P-36:6 PC 38:6 DG 38:5 PG 34:1; PGP-35:0 PC 38:5 DG 38:6 PG 34:2; PG P-35:1 PC 38:4 Hex2Cer d18:1/16:0;Hex2Cer PG 36:1; PG P-37:0 PC 38:3 d18:1/15:1 (1OH) Hex2Cer d18:1/20:0;Hex2Cer PG 36:2; PG P-37:1 PC 38:2 d18:1/19:1 (1OH) Hex2Cer d18:1/21:0(1OH) PG 36:3; PG P-37:2 PC 40:6 Hex2Cer d18:1/22:0; Hex2Cer PG 36:4; PGP-37:3 PC 40:5 d18:1/21:1 (1OH) Hex2Cer d18:1/24:1; Hex2Cer PI 32:0; PIO-33:0 PC 40:4 d18:1/23:2 (1OH) HexCer d18:1/16:0; HexCer PI 32:1; PIP-33:0 PC O-44:5/P-44:4 d18:1/15:1 (1OH) HexCer d18:1/20:0; PI 34:0; PIP-36:6 SM d34:2 HexCer d18:1/19:1 (1OH) HexCer d18:1/22:0; PI 34:1; PIP-35:0 SM d34:1 HexCer d18:1/21:1 (1OH) HexCer d18:1/24:0; PI 34:2; PIP-35:1 SM d36:2 HexCer d18:1/23:1 (1OH) HexCer d18:1/24:1; PI 36:0; PIP-38:6 SM d36:1 HexCer d18:1/23:2 (1OH) HexCer d18:1/25:0; PI 36:1; PIP-37:0 SM d38:2 HexCer d18:1/24:1 (1OH) LPC 16:0 PI 36:2; PI P-37:1 SMd38:1 LPC 16:1 PI 36:3; PI P-37:2 SM d39:1 LPC 18:0 PI 36:4; PI P-37:3SM d40:2 LPC 18:1 PI 38:2; PI P-40:8; PI P-39:1 SM d40:1 LPC 18:2 PI38:3; PI P-40:9; PI P-39:2 SM d41:2 LPC 20:1 PI 38:4; PI P-40:10; PIP-29:3 SM d41:1 LPC 20:3 PI 38:5; PI P-39:4 SM d42:3 LPC 20:4 PI 38:6;PI P-39:5 SM d42:2 LPC 22:6 PI 40:0; PI P-42:6, PI O-41:0 SM d42:1 LPE16:0 PI 40:4; PI P-42:10; PI P-4L3 TG 46:1 LPE 18:0 PI 40:5; PI P-41:4TG 46:0 LPE 18:1 PI 40:6; PI P-41:5 TG 47:1 LPE 18:2 PI 42:3; PI P-43:2TG 47:0 LPE 20:4 PI P-36:0; PI 35:1 TG 48:3 LPE 22:6 PI P-36:1; PI 35:2TG 48:2 LPG 20:0 PI P-38:3; PI 37:4 TG 48:1 LPG O-18:0; LPG 17:0 PS34:0; PS P-36:6 TG 48:0 LPG O-20:0; LPG 19:0 PS 34:2; PS P-35:1 TG 49:2LPG O-22:0; LPG 21:0 PS 36:1; PS P-37:0 TG 49:1 MG 16:0 PS 38:4; PSP-40:10; PS P-29:3 TG 50:4 MG 17:0 PS 38:6; PS P-39:5 TG 50:3 MG 18:0 PS42:1; PS P-43:0 TG 50:2 MG 18:1 PS P-32:0; PS-31:1 TG 50:1 MG 22:0 SM32:1 TG 51:6 PC 30:0 SM 33:1 TG 51:5 PC 32:0; PC O-33:0 SM 34:0 TG 51:4PC 32:1; PC P-33:0 SM 34:1 TG 51:3 PC 32:2; PC P-33:1 SM 34:2 TG 51:2 PC34:1; PC P-35:0 SM 35:1 TG 51:1 PC 34:2; PC P-35:1 SM 36:0 TG 52:6 PC34:3; PC P-35:2 SM 36:1 TG 52:5 PC 34:4; PC P-35:3 SM 36:2 TG 52:4 PC36:0; PC P-38:6 SM 37:0 TG 52:3 PC 36:1; PC P-37:0 SM 39:1 TG 52:2 PC36:2; PC P-37:1 SM 40:0 TG 52:1 PC 36:3; PC P-37:2 SM 40:1 TG 53:4 PC36:4; PC P-37:3 SM 41:1 TG 53:3 PC 36:5; PC P-37:4 SM 41:2 TG 53:2 PC38:2; PC P-40:8; PC P-39:1 SM 42:2 TG 53:1 PC 38:3; PC P-40:9; PC P-39:2SM 43:1 TG 54:7 PC 38:4; PC P-40:10; PC P-29:3 SM 44:2 TG 54:6 PC 38:5;PC P-39:4 Sulfatide 41:0; Sulfatide 40:1 (1OH) TG 54:5 PC 38:6; PCP-39:5 Sulfatide 41:1; Sulfatide 40:2 (1OH) TG 54:4 PC 40:4; PC P-42:10;PC P-41:3 Sulfatide 43:0; Sulfatide 42:1 (1OH) TG 54:3 PC 40:5; PCP-41:4 Sulfatide 43:1; Sulfatide 42:2 (1OH) TG 54:2 PC 40:6; PC P-41:5Sulfatide 43:2; Sulfatide 42:3 (1OH) TG 55:6 PC O-32:0; PC-31:0 TG 56:3TG 55:4 PC O-34:0; PC 33:0 TG 56:4 TG 55:3 PC P-32:0; PC-31:1 TG 56:5 TG56:9 PC P-34:0; PC 33:1 TG 56:6 TG 56:8 PC P-34:1; PC 33:2 TG 58:6 TG56:7 PC P-36:0; PC 35:1 TG 56:6 PC P-36:1; PC 35:2 TG 56:5 PC P-36:2; PC35:3 TG 56:4 PC P-36:3; PC 35:4 TG 56:3 PC P-36:4; PC 35:5 TG 56:1 PCP-38:2; PC 37:3 TG 58:10 PC P-38:3; PC 37:4 TG 58:9 PC P-38:4; PC 37:5TG 58:8 PC P-38:5; PC 37:6 TG 58:7 PC P-40:3; PC 39:4 TG 58:6 PC P-40:4;PC 39:5 TG 60:11 PC P-40:5; PC 39:6 TG 60:10 230 121

BRIEF DESCRIPTION OF DRAWINGS

In the following descriptions, T1, T2, T3, T4, Tis, and Tx are stages ofcancer (primary tumor) according to TNM classification. T1 is a veryearly stage of cancer, Tis means carcinoma insitu, and Tx means thattumour cannot be assessed.

FIG. 1: PCA statistical model of 170 plasma samples of healthy controls(N), 282 plasma samples of patients suffering from cancer (T), 38 plasmasamples of unknown classification (X) and quality control (Q) for bothgenders (generated from UHPSFC/MS data).

FIG. 2: OPLS-DA statistical model of healthy control (N) and cancerousplasma samples (tumor stages T1, T2, T3, T4, Tis, and Tx) for bothgenders (generated from UHPSFC/MS data).

FIG. 3: S-plot representing the most up and down regulated lipid speciesfor comparison of non-cancerous and cancerous plasma samples.

FIG. 4: OPLS-DA statistical model of plasma samples of healthy controls(N) and patients suffering from breast, kidney, and prostate cancers(tumor stages T1, T2, T3, T4, and Tx) for males (generated fromUHPSFC/MS data).

FIG. 5: OPLS-DA statistical model of plasma samples of healthy controls(N) and patients suffering from breast cancer (T) for males (generatedfrom UHPSFC/MS data).

FIG. 6: OPLS-DA statistical model of plasma samples of healthy controls(N) and patients suffering from prostate cancer (tumor stages T2, T3,Tx) for males (generated from UHPSFC data).

FIG. 7: OPLS-DA statistical model of plasma samples of healthy controls(N) and patients suffering from kidney cancer (tumor stages T1, T2, T3,and Tx) for males (generated from UHPSFC data).

FIG. 8: OPLS-DA statistical model of plasma samples of healthy controls(N) and patients suffering from breast and kidney cancer types (tumorstages T1, T2, T3, T4, and Tx) for females (generated from UHPSFC/MSdata).

FIG. 9: OPLS-DA statistical model of plasma samples of healthy controls(N) and patients suffering from breast cancer (tumor stages T1, T2, Tis,and Tx) for females (generated from UHPSFC/MS data).

FIG. 10: OPLS-DA statistical model of plasma samples of healthy controls(N) and patients suffering from kidney cancer (tumor stages T1, T2, T3,and Tx) for females (generated from UHPSFC data).

FIG. 11: PCA statistical model of 170 samples of healthy controls (N),111 samples of patients suffering from kidney cancer (T) and 24 samplesof quality control pooled samples (Q) for both genders (generated fromMALDI-MS data).

FIG. 12: OPLS-DA statistical model of 170 samples of healthy controls(N), 111 samples of patients suffering from kidney cancer (tumor stagesT1, T2, T3, and Tx) for both genders (generated from MALDI-MS data).

FIG. 13: OPLS-DA analysis of 75 samples of healthy controls (N), 80samples of patients suffering from kidney cancer (tumor stages T1, T2,T3, and Tx) for males (generated from MALDI-MS data).

FIG. 14: OPLS-DA analysis of 95 samples of healthy controls (N), 32samples of patients suffering from kidney cancer (tumor stages T1, T2,T3, and Tx) for females (generated from MALDI-MS data).

FIG. 15: PCA analysis of 70 samples of healthy controls (N), 101 samplesof patients suffering from kidney cancer (T) and 24 samples of qualitycontrol pooled samples (Q) for both genders (generated from MALDI-MSdata).

FIG. 16: OPLS-DA analysis of 70 samples of healthy controls (N), 101samples of patients suffering from kidney cancer (tumor stages T1, T2,T3, and Tx) for both genders (generated from MALDI-MS data).

FIG. 17: OPLS-DA analysis of 34 samples of healthy controls (N), 72samples of patients suffering from kidney cancer (tumor stages T1, T2,T3, and Tx) for males (generated from MALDI-MS data).

FIG. 18: OPLS-DA analysis of 36 samples of healthy controls (N), 29samples of patients suffering from kidney cancer (tumor stages T1, T2,T3, and Tx) for females (generated from MALDI-MS data).

FIG. 19: OPLS-DA statistical model of breast, kidney, and prostatecancer plasma samples from males.

FIG. 20: OPLS-DA plot statistical model for the differentiation ofprostate and kidney cancer plasma samples from males.

FIG. 21: OPLS-DA statistical model for the differentiation of breast andkidney cancer plasma samples from females.

FIG. 22. OPLS-DA analysis of 79 samples of healthy controls (N), 213samples of patients suffering from pancreatic cancer (tumor stages T1,T2, T3, T4, and Tx) for both genders (generated from UHPSFC/MS andshotgun data).

FIG. 23. OPLS-DA analysis of 49 samples of healthy controls (N), 109samples of patients suffering from pancreatic cancer (tumor stages T1,T2, T3, T4, and Tx) for males (generated from UHPSFC/MS and shotgundata).

FIG. 24. OPLS-DA analysis of 30 samples of healthy controls (N), 104samples of patients suffering from pancreatic cancer (tumor stages T1,T2, T3, T4, and Tx) for females (generated from UHPSFC/MS and shotgundata).

FIG. 25. OPLS-DA statistical model prediction of being pancreatic cancerpatient for females from UHPSFC/MS and shotgun data for unknown samples.

FIG. 26. OPLS-DA statistical model prediction of being pancreatic cancerpatient for males from UHPSFC/MS and shotgun data for unknown samples.

DETAILED DESCRIPTION OF THE INVENTION

Sample Collection:

Human body fluid samples of cancer patients and healthy volunteers arecollected in the hospital, typically blood or urine. Other samples typesare isolated from blood, such as plasma, serum, oncosomes, exosomes,extracellular vesicles, etc. The blood of human subjects is collected inthe standard and well established way used in hospitals intoanticoagulant-containing tubes, such as EDTA, heparin, citrate tubes,then serum or plasma is isolated using standardized protocols well knownto a person skilled in the art, storage at −80° C. at the clinic,transport from the clinic to the analytical laboratory using biologicaltransport bags with dry ice (−20° C.), storage again at −80° C. untilthe analysis.

Preparation of a Set of Internal Standards (Mixture of InternalStandards):

Generally, an internal standard mixture is used for quantitation oflipid species. The internal standard for each lipid class behaves in thesame way as the target compounds belonging to this lipid class. Due tothe addition of the internal standard before sample processing, slightdifferences due to for instance pipetting errors can be compensated asthe internal standard is affected in the same way as the targetcompounds.

In a particular embodiment, 2-4 mg of each standard is weighed into 2 mLHPLC glass vials using an analytical balance and dissolved incorresponding volume (chloroform:2-propanol—2:8) in order to obtain afinal concentration of 2, 2.1, 1 or 0.25 μg/μL. Standards for each lipidclass are mixed together to form internal standard mixtures as describedin Table 16 for all herein shown types of MS analysis. Preferably, onemixture of internal standards is prepared in a sufficient amount for thewhole experiment, in order to avoid variances in quantitation due toslight differences in concentration of the internal standards when themixture is prepared in several batches. Aliquots may be prepared of thisinternal standard mixture and stored at −80° C.

TABLE 16 Preparation of internal standards mixture. Stock Concentrationconcentration Volume in plasma Internal standards MW [μg/μL] [μL][nmol/mL] CE d7 16:0 631.6285 2 200 443.3 Cer d18:1/12:0 481.4495 2 411.6 DG 12:1/12:1 452.3502 2 15 46.4 Hex2Cer d18:1/12:0 805.5551 2 4 7.0HexCer d18:1/12:0 643.5023 2.1 3 6.9 Chol d7 393.6988 2 300 1066.8 PI33:1 d7 846.596 1 16 13.2 LPC 17:0 509.3481 2.1 20 57.7 LPE 14:0425.2542 2 4 13.2 LPG 14:0 478.2308 2.1 14 43.0 MG 19:1 370.3083 2 30113.4 PA 14:0/14:0 614.3924 2 4 9.1 PC 14:0/14:0 677.4996 2 60 124.0 PE14:0/14:0 635.4526 2 4 8.8 PG 14:0/14:0 688.4291 2 2 4.1 PS 14:0/14:0701.4244 2 4 8.0 SM d18:1/12:0 646.505 2 20 43.3 SulfoHexCer d18:1/12:0740.4857 0.25 0.28 0.1 TG 19:1/19:1/19:1 926.8302 2 75 113.3 PC 44:2897.719 2 80 124.8 Solvent CHCl₃:IPA 2:8 140.72 Total Volume 1000

Sample Processing:

All samples are spiked before the extraction with the appropriateinternal standard mixture (depending on which body fluid and which massspectrometry method is used).

The lipidomic extractions were preferably performed by well-establishedprocedures using chloroform-methanol-water extraction systems aspublished in our previous works [E. Cífková, M. Holčapek, M. Lísa, D.Vrána, J. Gatěk, B. Melichar, Anal. Bioanal. Chem. 407 (2015) 991-1002;E. Cífková, M. Lísa, R. Hrstka, D. Vrána, J. Gatěk, B. Melichar, M.Holčapek, Rapid Commun. Mass Spectrom. 31 (2017) 253-263]. The order ofsamples for extractions is randomized. 25 μL of serum or plasma and 17.5μL of internal standard mixture and 2 mL chloroform (2×1 mL) and 1 mLmethanol are transferred into a glass vial (4 mL). Biological samplescontain also proteins, which may lead to sticky and slimy samples. Itmust be ensured that the pipette draws the 25 μL of serum or plasma andis not blocked with the slimy components during drawing. The glass vialsare closed and put for 10 min into the ultrasonic bath at 40° C. forhomogenization. Afterwards, the samples are allowed to reach roomtemperature in order to ensure no evaporation of the organic solventswhen opening the vials. 600 μL of water are added to each sample. Thesamples are closed and vortexed for 1 min. It is important to do theextraction step always in the same way for all samples of one batch (onestudy). The target lipids for mass spectrometry are better soluble inthe organic layer (bottom layer). Afterwards, the samples arecentrifuged for 3 min at 3000 rpm in order to separate the organic andaqueous layer (in between these two layers a protein layer in the formof white precipitate is formed). The aqueous layer (upper layer) isremoved via a glass pipette. The organic solvent containing the targetcompounds is evaporated under a stream of nitrogen at 30° C. and theglass vials containing the residue (target compounds) are stored at −80°C.

Before analysis, the samples are allowed to reach ambient temperature(so that they cannot draw water from the air when opening the vials) andthen the residue from the extraction and evaporation (previous step) isdissolved in 500 μL chloroform:2-propanol (1:1, v/v). It is advisable toprepare enough solvent mixture of chloroform:2-propanol (1:1, v/v)mixture for all samples to avoid variations between individual batchesof the solvent mixture. The samples are vortexed carefully for 1 min toensure that the target compounds are dissolved. The solution is filteredusing a 0.2 μm syringe filter in order to get rid of undissolvedcomponents, which may compromise the MS analysis. The vials are closedwith PTFE caps and stored at −80° C.

For urine samples, reversed-phase solid phase extraction is performed. 2mL of human urine together with 3 μL of mixture of internal standardsdissolved in methanol (SufoHexCer d18:1/12:0 of concentration 1.7 μg/mLand D4 taurocholic acid of concentration 16.7 μg/mL) are loaded on 200mg tC18 cartridge (Sep-Pak Vac, 37-55 μm particle size) (Waters,Milford, Mass., USA) previously primed with 3 mL of methanol followed by3 mL of water. Columns are washed with 3 mL of water, and studied lipidsare further eluted with 3 mL of methanol. The eluates are collected,then evaporated by the gentle stream of nitrogen and redissolved in themixture of 300 μL of methanol before the measurement.

Sample Preparation for UHPSFC/MS Analysis

The filtrate is diluted 5 times or 20 times with a mixture ofhexane:2-propanol:chloroform 7:1.5:1.5 and transferred into an HPLCvial. The vials containing the diluted filtrate are closed with slitcaps for analysis with UHPSFC/MS and placed in the autosampler.

Sample preparation for shotgun MS analysis The filtrates are diluted 10times depending on the samples by chloroform-methanol-2-propanol (1:2:4,v/v/v) mixture containing 7.5 mmol/L of ammonium acetate and 1% ofacetic acid.

Sample preparation for MALDI-MS analysis

MALDI matrix 9-aminoacridine (Sigma-Aldrich, St. Louis, Mo., USA) isdissolved in methanol-water mixture (4:1, v/v) to provide theconcentration of 5 mg/ml or 10 mg/ml and mixed with particular lipidextracts (1:1, v/v)—less preferably lipid extracts can be diluted withmethanol (1:1, 1:2, or 1:3, v/v) before mixing with matrix. Thedeposited amount of extract/matrix mixture is 1 μl and the dried dropletcrystallization is used for the sample deposition on the target plate.The deposition of small aliquot of chloroform on MALDI plate spotsbefore the application of diluted extract/matrix mixture is applied toavoid the drop spreading.

Mass Spectrometry (MS) Method Development and Validation

Three major MS based methods for lipidomic quantitation were developedin particular, which are described in more detail here. The methodbenefits from the use of pooled sample, which is a mixture of identicalvolumes of all samples for smaller studies with less than 100 subjects.In this study, the pooled sample is prepared from equal volumes ofrandomly selected cancer patients and healthy volunteers samples,keeping the ratio of males and females in the same proportion as in thesample set. The pooled sample is used for the method development andoptimization. The pooled sample with added internal standard mixture pereach lipid class to be quantified is used for the full validation and QCduring the measurements. The order of samples is always randomized insample sequences to avoid measurements of non-cancerous and canceroussamples in certain portion of sequence.

The system suitability test was carried out before the validationprocedure at three concentration levels typically reported as low,medium and high concentration levels. All concentration levels must bewithin the linear dynamic range. The low concentration level is close tothe lower limit of quantitation (LLOQ), the middle concentration levelis in the middle of the linear dynamic range, and the high concentrationlevel is close to the upper limit of quantitation (ULOQ). In aparticular embodiment, we use 5, 17.5, and 30 μl of IS mixture preparedaccording to Table 16 for low, medium and high concentration levels,respectively. Validation parameters such as selectivity, accuracy,precision, calibration curve, limits of detection and quantitation,matrix effect, carry-over and stability were determined. Individualparameters were determined for IS representing properties of the lipidclass. The selectivity was determined using 3 extracts of the pooledserum sample spiked before extraction with the IS mixture at low, middleand high concentration level and 3-6 extracts of appropriate non-spikedserum samples. The accuracy and precision were studied using the pooledserum sample spiked after the extraction at low, medium and highconcentration levels. The intra-day accuracy and intra-day precisionwere studied using three samples per concentration level. The inter-dayaccuracy and inter-day precision were evaluated among three independentruns on two different days using three samples at the low, medium andhigh concentration level. The LLOQ and ULOQ corresponded to the firstand the last points of linearity range, respectively.

The limit of detection (LOD) was determined based on signal to noiseratio (S/N=3) observed from reconstructed ion chromatogram or neutralloss (NL) and precursor ion (PI) mass spectra (shotgun MS) of internalstandard mixture. The extraction recovery was determined by calculatingthe ratio of the signal response of samples spiked before and afterextraction for low, medium and high concentration. The processefficiency was determined by calculating the ratio of the signalresponse of the spiked samples before extraction and the neat standardat different concentrations. The matrix effect was calculated from theratio of the signal response of samples spiked after extraction and theneat standard. The carry-over was evaluated for each IS by the injectionof blank sample with the pure solvent after the calibration sample athigh concentration level (dilution factor of 10). The reliability ofresults obtained within analysis of large sample sets was evaluated byon-instrument and freeze-and-thaw stability tests. The stability ofspiked plasma extract at middle concentration level was measured inautosampler at certain time intervals: 0, 4, 8, 12, 16, and 24 hours.Sample for freeze-and-thaw experiment was analyzed immediately aftercomplete unassisted thawing in autosampler.

TABLE 17 Dilution scheme for calibration with pooled sample forUHPSFC/MS (volumes in μl) for plasma samples. Hexane: 2-propanol:Mixture Pooled chloroform of IS sample (7:1.5:1.5, v/v/v) 150 100 250100 100 300  50 100 350  20 100 380  15 100 385  10 100 390  5 100 395  3.5 100   396.5  2 100 398 IS mixture 1: 50 diluted  75 100 325  50100 350  25 100 375  10 100 390   7.5 100   392.5  5 100 395   2.5 100  397.5  1 100 399

For calibration, the optimized mixture of IS for all methods in severaldilutions were used in order to calibrate in the concentration rangesrelevant for individual lipid classes. Depending on the dilution factorof the corresponding mass spectrometric method, the correspondingamounts of matrix were used. For instance, UHPSFC/MS uses 1:5 dilutionof sample extracts. Therefore, 1/5 of blank plasma of a pooled samplewere added (see Table 17 for more details).

UHPSFC/MS

Supercritical fluid chromatography is a tool for the separation ofcompounds of different polarity employing supercritical carbon dioxide(mobile phase) as main component for removing the compounds from anadsorbent (column-stationary phase). The addition of an organic solvent(typically methanol) to the supercritical carbon dioxide broadens theapplication range of UHPSFC and allows the removal of more polarcompounds from the column. Generally, compounds can be differentiated ifthey are better soluble in water or alcohols, than they are of polarnature or if they are better soluble for instance in hexane than theyare of nonpolar nature. Depending on the nature of the stationary phase,the mobile phase and the target compounds, like polar or nonpolar,interactions can be forced. For instance, polar compounds prefer tointeract with polar stationary phases and in order to remove those fromthe stationary phase, a polar mobile phase can be used to remove thecompounds from the stationary phase. A careful adjustment of theseinteractions by optimization of the mobile phase properties using acertain stationary phase allows the separation of compounds. Theoptimization of dimensions and properties of the stationary phase, suchas smaller particle size and spherical particles of the sorbents, allowsfor a higher efficiency and is therefore called ultrahigh performancesupercritical fluid chromatography (UHPSFC).

The chromatographic separation can be optimized for the separation oflipid classes, taking into account in particular the following. A lipidclass has a dominant structural moiety (polar head group) in commonwhich is mainly responsible for the interaction governing the retentionmechanism. A lipid class can comprise numerous lipid species varying inthe hydrocarbon chain length and structure (e.g., double bonds).Internal standards are added for each lipid (sub)class, therefore it ispossible to identify and quantify all lipid species within a particularlipid (sub)class by comparing it to the class internal standard.MS-Analysis: Using a high-resolution, accurate-mass spectrometer as adetector allows the identification and quantification of lipids, as eachlipid species has a defined m/z value and gives a signal responsedepending on the concentration in the sample. In order to improveionization and therefore the signal response of the target compounds,additives like acids and buffers are added to the sample or to themobile phase. In case of UHPSFC/MS, the use of a make-up solvent likeacidified methanol further improves the sensitivity.

In one particular embodiment, a detailed description of all parametersapplied for the UHPSFC/MS method to obtain the results presented hereinbelow is as follows: Instrument-Acquity Ultra Performance ConvergenceChromatography (UPC²) System hyphenated to the hybridquadrupole-traveling wave ion mobility-time of flight mass spectrometerSynapt G2 Si from Waters. Chromatographic settings-stationary phase:Acquity BEH UPC² column (100×3 mm, 1.7 μm, Waters), the flow rate was1.9 mL/min, the injection volume 1 μL, the column temperature 60° C.,the active back pressure regulator (ABPR) was set to 1800 psi, gradientmode:CO₂ and methanol with 30 mM ammonium acetate and 1% water. Thegradient started at 1% modifier and increased to 51% in 5 mM, afterwardskept constant for 1 mM and flushed back to starting conditions with atotal run time of 7.5 mM. injection needle wash: a mixture ofhexane-2-propanol-water (1:2:0.5, v/v) column wash after each biologicalsample injection: a blank was injected using a fast gradient: 0 mM-1%,1.4 mM-51%, 1.6 mM-51%, 1.8 mM-1%, and 4.8 mM-1% modifier, make-upeffluent: HPLC 515 pump (Waters), make-up flow rate 0.25 mL/min methanolwith 1% water, and optionally 0.1% formic acid.

ESI-MS settings: a capillary voltage of 3 kV, a sampling cone of 20 V,the source offset of 90 V, a source temperature of 150° C., a dryingtemperature of 500° C., the cone gas flow of 50 L h⁻¹, the drying gasflow of 1000 L h⁻¹ and the nebulizer gas of 4 bar. Resolution mode orsensitivity mode in positive ion mode and a mass range of m/z 50-1200.The scan time was 0.15 s, and measurements were performed in continuummode. The peptide leucine enkephaline was used as the lock mass with ascan time of 0.1 s and interval of 30 s.

During the analysis, the samples in the sequence should be randomized sothat not the same type of samples, such as only healthy (non-cancerous)samples, are measured in a row. This guarantees that in case of an errorat a certain time not only one type of sample is affected. Furthermore,it is important to measure QC samples after a predetermined amount ofsamples in order to verify the instrument performance.

Before measuring biological samples, no injection, blank and QC samplesare measured to check the instrument performance and afterwards it iscontinued with biological samples. All samples are measured induplicates and after 20 samples or 40+40 injections (sample+wash)respectively, QC and blank samples are measured. During the whole study,measurement and sample preparation control is performed by evaluatingthe peak areas of each internal standard of each sample and exportingresults in the Microsoft Excel file. The QC samples are aliquots of anextract of a mixture of serum or plasma samples. The lock mass iscontinuously measured during analysis, however the lock mass correctionis not applied online, as preliminary results showed that the massaccuracy is worse using online correction. Furthermore, continuum modeis applied so that it is possible to monitor the resolution of theinstrument. After measurements, the raw data get noise reduced using theMassLynx software from Waters. This improves the mass spectra as well assignificantly reduces the file size, which allows easier handling of thefiles for further processing. The files are further processed byapplying the lock mass correction and converting the files from profileto centroid mode, which enhances the mass accuracy and further reducesthe file size. (The file size is important for data processing. Theprocessing software can only hardly deal with huge file sizes and samplenumbers, resulting in continuous errors making the processingtime-consuming and cumbersome.)

All investigations regarding measurement control, profile mode andoffline lock mass correction as well as the noise reduction improveddata quality.

Shotgun MS

Experiments as presented herein below were performed on aquadrupole-linear ion trap mass spectrometer 6500 QTRAP (Sciex, Concord,ON, Canada) equipped by ESI probe with the following setting of tuningparameters: the ionspray voltage 5200 V, the curtain gas 20 psi, thesource temperature 50° C., the ion source gas(1) 15 psi, and the ionsource gas(2) 10 psi. MS/MS scans are measured with the scan rate 1000Da/s, the declustering potential 80 V, the entrance potential 10 V, andthe collision energy specified in the Table 18. Samples are introducedby a flow injection using a liquid chromatograph Agilent 1290 Series(Agilent Technologies) consisted of Agilent 1290 binary pump and Agilent1260 autosampler. 50 μL of sample was injected into the flow rate 3μL/min of chloroform-methanol-2-propanol (1:2:4, v/v/v) mixturecontaining 7.5 mmol/L of ammonium acetate and 1% of acetic acid with theanalysis time 12 min, and the autosampler temperature 20° C. LC/MSsystem is washed after each analysis with methanol-2-propanol-water(2:2:1, v/v/v) mixture containing 7.5 mmol/L of ammonium acetate and 1%of acetic acid. Measured data experiments are extracted using LipidViewsoftware with the mass tolerance 0.3 Da, the minimum S/N=5 and theminimum intensity 1%. Raw data characterized by type of scans, m/zvalues and peak intensities are exported as .txt data and furtherprocessed using our

Microsoft Excel macro script for the detection and quantitation oflipids. Lipid classes are characterized using type of scans, andindividual lipid species in selected MS/MS scan are detected accordingto m/z values with the mass tolerance 0.3 Da based on the databasecompiled from identified lipids in the pooled sample followed by theisotopic correction of ion intensities. Concentration of lipid speciesare calculated from corrected ion intensity related to the intensity oflipid class internal standards.

TABLE 18 Characterization of shotgun MS/MS scans and their parametersfor individual lipid classes. PI—precursor ion scan and NL—neutral lossscan. Type of MS/MS Collision Observed Lipid class scan energy ions CEPI 369; PI 376 12 [M + NH₄]⁺ Cer PI 264; PI 266 35 [M + H]⁺ DG NL 35 20[M + NH₄]⁺ Hex2Cer PI 264; PI 266 35 [M + H]⁺ HexCer PI 264; PI 266 35[M + H]⁺ Cholesterol PI 369; PI 376 12 [M + NH₄]⁺ LPA NL 115 25 [M +NH₄]⁺ LPC PI 184 35 [M + H]⁺ LPE NL 141 30 [M + H]⁺ LPG NL 189 30 [M +NH₄]⁺ LPS NL 185 30 [M + H]⁺ MG NL 35 20 [M + NH₄]⁺ PA NL 115 25 [M +NH₄]⁺ PC PI 184 35 [M + H]⁺ PE NL 141 30 [M + H]⁺ PG NL 189 30 [M +NH₄]⁺ PS NL 185 30 [M + H]⁺ SM PI 184 35 [M + H]⁺ SulfoHexCer NL 98 25[M + H]⁺ TG NL 17 30 [M + NH₄]⁺

MALDI Mass Spectrometry

Mass spectra were measured using ultrahigh-resolution MALDI massspectrometer LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, Mass.,USA) equipped with the nitrogen UV laser (337 nm, 60 Hz) with a beamdiameter of about 80 μm×100 μm. The LTQ Orbitrap instrument is operatedin the negative-ion mode over a normal mass range m/z 400-2000 and themass resolution is set to R=100,000 (full width at half maximumdefinition, at m/z 400). The zig-zag (or spiral outwards) samplemovement with 250 μm step size is used during the individual dataacquisition. The laser energy corresponds to 15% of maximum and 2microscans/scan with 2 laser shots per microscan at 36 differentpositions are accumulated for each measurement to achieve a reproduciblesignal. Each sample (spotted matrix and body fluid extract mixture) isspotted five times. The total acquisition time of one sample includingfive consecutive spots is around ten minutes. Each measurement isrepresented by one average MALDI-MS spectrum with thousands of m/zvalues. The automatic peak assignment is subsequently performed andparticular m/z peaks are matched with deprotonated molecules from adatabase created during the identification procedure using the Excelmacro script. This peak assignment results in the generation of the listof present m/z of studied lipids with the average intensities inparticular spectra for each samples that is used for further statisticalevaluation.

Data Processing and Quantitation

The data processing starts with the data export from MS vendor software(e.g., Waters, Sciex or Thermo Scientific) into a data-processingsoftware which may be Microsoft Excel for further steps to be donesemi-automatically using e.g. advanced Excel script, in particularisotopic correction (Tables 19 and 20) and zero-filling. Quality control(QC) samples should be regularly injected to check the right andconstant response of mass spectrometer. The typical QC sample is apooled sample containing internal standards for all lipid classes to bequantified, which is injected after every 20 injections, and responsesof individual internal standards are plotted versus the time. Ifresponses of the internal standards are reduced too much, then it is theindication of an instrumental problem, typically the mass spectrometerrequires cleaning due to the injection of too many samples. The typicalcleaning interval is about several hundreds of samples, but it maystrongly depend on the quality of prepared sample extracts, geometry ofion source, and system configuration.

The following example describes the example of UHPSFC/MS measurement onSynapt G2Si instrument from Waters, but similar approach is alsoapplicable for other MS methods and different MS platforms from anyinstrumental vendor. The noise reduced, lock mass corrected andconverted files are further processed with the MarkerLynx software. Thereduced sequence table only including serum or plasma samples and QCsamples has to be prepared in MassLynx with the corresponding suffix inthe sample name (sample_nr20_AFAMM). Then the time scan range for eachlipid class over the whole sequence has to be determined (for examplem/z 250-350 is used for CE). For each lipid class, the method is createdin MarkerLynx with the corresponding scan range, which will be combined,the mass peak separation of 50 mDa and marker intensity threshold of3000. The method for each lipid class is applied for the sequence andMarkerLynx table with m/z values against the combined intensities iscreated. This table is exported into a text file and imported into ahomemade database for the identification and quantitation of lipidspecies using Microsoft Excel. The m/z values obtained from MarkerLynxare compared to the accurate m/z values deposited as database forhundreds of lipid species for the identification. The database wascreated by evaluating present species in tissue and plasma samples. Theidentified lipid species are isotopically corrected and quantified bycalculating the concentration in relation to the intensity andconcentration of the (sub)class IS obtaining the table of lipid speciesconcentrations vs. individual samples. Zero-filling procedure isperformed. The average of the multiple injected samples for individuallipid species is calculated. The final data matrix, where the columnsare individual subjects and lines are individual lipids, are used forfurther MDA statistical evaluation for absolute quantification. Forrelative quantification, the concentration of individual species withina class is related to the sum of concentrations of this lipid class inone sample. The resulting table is then used for statistical evaluationequally as for the absolute quantification.

TABLE 19 Database of lipid species monitored during the identificationstep in the positive-ion mode together with the calculation of isotopiccorrection for M + 1 and M + 2 isotopic peaks (so called deisotoping).Isotopic correction Lipid m/z M + 2 M + 1 CE 16:0 D7 (IS) 376.3955 0.00%0.00% CE 10:0 558.5244 0.00% 0.00% CE 12:0 586.5557 0.00% 0.00% CE 14:1612.5714 0.00% 0.00% CE 14:0 614.5870 10.59% 0.00% CE 15:1 626.55320.00% 0.00% CE 15:0 628.6027 11.09% 0.00% CE 16:1 640.6027 0.00% 0.00%CE 16:0 642.6184 11.61% 0.00% CE 16:0 D7 (IS) 649.6623 0.00% 0.00% CE17:1 654.6183 0.00% 0.00% CE 17:0 656.6340 12.14% 0.00% CE 18:4 662.58700.00% 0.00% CE 18:3 664.6027 12.64% 0.00% CE 18:2 666.6184 12.65% 0.00%CE 18:1 668.6340 12.67% 0.00% CE 18:0 670.6497 12.68% 0.00% CE 19:1682.6497 0.00% 0.00% CE 19:0 684.6653 13.23% 0.00% CE 20:5 688.60270.00% 0.00% CE 20:4 690.6184 13.75% 0.00% CE 20:3 692.6340 13.76% 0.00%CE 20:2 694.6497 13.77% 0.00% CE 20:1 696.6653 13.78% 0.00% CE 20:0698.6810 13.80% 0.00% CE 21:1 710.6809 0.00% 0.00% CE 21:0 712.696614.37% 0.00% CE 22:6 714.6184 14.39% 0.00% CE 22:5 716.6340 14.90% 0.00%CE 22:4 718.6496 14.91% 0.00% CE 22:3 720.6653 14.93% 0.00% CE 22:2722.6810 14.94% 0.00% CE 22:1 724.6966 14.95% 0.00% CE 22:0 726.712314.96% 0.00% CE 23:1 738.7122 0.00% 0.00% CE 23:0 740.7279 15.57% 0.00%CE 24:5 744.6653 0.00% 0.00% CE 24:4 746.6809 16.13% 0.00% CE 24:3748.6966 16.14% 0.00% CE 24:2 750.7122 16.15% 0.00% CE 24:1 752.72790.00% 0.00% CE 24:0 754.7436 16.18% 0.00% CE 25:0 768.7592 0.00% 0.00%CE 26:5 772.6966 0.00% 0.00% CE 26:4 774.7122 17.39% 0.00% CE 26:3776.7279 17.40% 0.00% CE 26:0 782.7748 0.00% 0.00% CE 27:0 796.79050.00% 0.00% CE 28:0 810.8061 0.00% 0.00% TG 35:0 642.5667 0.00% 0.00% TG36:1 654.5667 0.00% 0.00% TG 36:0 656.5823 10.53% 0.00% TG 38:3 678.56670.00% 0.00% TG 38:2 680.5823 11.49% 0.00% TG 38:1 682.5980 11.50% 0.00%TG 38:0 684.6136 11.51% 0.00% TG 40:4 704.5823 0.00% 0.00% TG 40:3706.5980 12.50% 0.00% TG 40:2 708.6136 12.51% 0.00% TG 40:1 710.629312.52% 0.00% TG 40:0 712.6449 12.54% 0.00% TG 41:1 724.6449 0.00% 0.00%TG 41:0 726.6606 13.07% 0.00% TG 42:4 732.6136 0.00% 0.00% TG 42:3734.6293 0.00% 0.00% TG 42:2 736.6449 13.59% 0.00% TG 42:1 738.660613.60% 0.00% TG 42:0 740.6763 13.61% 0.00% TG 43:1 752.6763 0.00% 0.00%TG 43:0 754.6919 14.17% 0.00% TG 44:4 760.6449 0.00% 0.00% TG 44:3762.6606 0.00% 0.00% TG 44:2 764.6763 14.71% 0.00% TG 44:1 766.691914.72% 0.00% TG 44:0 768.7076 14.73% 0.00% TG O-46:5/P-46:4 772.68130.00% 0.00% TG O-46:4/P-46:3 774.6970 15.64% 0.00% TG 45:3 776.67620.00% 0.00% TG 45:2 778.6919 15.29% 0.00% TG 45:1 780.7076 15.30% 0.00%TG 45:0 782.7232 15.31% 0.00% TG 46:4 788.6763 0.00% 0.00% TG 46:3790.6919 15.87% 0.00% TG 46:2 792.7076 15.58% 0.00% TG 46:1 794.723215.89% 0.00% TG 46:0 796.7389 15.91% 0.00% TG 47:3 804.7076 0.00% 0.00%TG 47:2 806.7232 16.48% 0.00% TG 47:1 808.7389 16.50% 0.00% TG 47:0810.7545 16.51% 0.00% TG 48:5 814.6919 0.00% 0.00% TG 48:4 816.70760.00% 0.00% TG 48:3 818.7232 17.09% 0.00% TG 48:2 820.7389 17.10% 0.00%TG 48:1 822.7545 17.11% 0.00% TG 48:0 824.7702 17.13% 0.00% TG 49:3832.7389 0.00% 0.00% TG 49:2 834.7545 17.73% 0.00% TG 49:1 836.770217.74% 0.00% TG 49:0 838.7858 17.76% 0.00% TG 50:6 840.7076 18.32% 0.00%TG 50:5 842.7232 18.33% 0.00% TG 50:4 844.7389 18.34% 0.00% TG 50:3846.7545 18.36% 0.00% TG 50:2 848.7702 18.37% 0.00% TG 50:1 850.785818.38% 0.00% TG 50:0 852.8015 18.40% 0.00% TG 51:6 854.7232 18.41% 0.00%TG 51:5 856.7389 18.98% 0.00% TG 51:4 858.7545 18.99% 0.00% TG 51:3860.7702 19.01% 0.00% TG 51:2 862.7858 19.02% 0.00% TG 51:1; TG 52:8864.8015 19.04% 0.00% TG 52:7 866.7232 0.00% 0.00% TG 51:0 866.817119.04% 0.00% TG 52:6 868.7389 19.06% 0.00% TG 52:5 870.7545 19.65% 0.00%TG 52:4 872.7702 19.66% 0.00% TG 52:3 874.7858 19.67% 0.00% TG 52:2876.8015 19.69% 0.00% TG 52:1 878.8171 19.70% 0.00% TG 52:0 880.832819.72% 0.00% TG 53:6 882.7545 19.73% 0.00% TG 53:5 884.7702 20.32% 0.00%TG 53:4 886.7858 20.34% 0.00% TG 53:3; TG 54:10 888.8015 20.35% 0.00% TG53:2; TG 54:9 890.8171 20.36% 0.00% TG 54:8 892.7388 0.00% 0.00% TG 53:1892.8327 20.38% 0.00% TG 54:7 894.7545 20.98% 0.00% TG 53:0 894.848420.39% 0.00% TG 54:6 896.7702 20.41% 0.00% TG 54:5 898.7858 21.01% 0.00%TG 54:4 900.8015 21.02% 0.00% TG 54:3 902.8171 21.04% 0.00% TG 54:2904.8328 21.05% 0.00% TG 54:1 906.8484 21.07% 0.00% TG 54:0 908.864121.08% 0.00% TG 55:6 910.7858 21.10% 0.00% TG 55:5 912.8015 21.71% 0.00%TG 55:4 914.8171 21.73% 0.00% TG 55:3; TG 56:10 916.8328 21.74% 0.00% TG56:9 918.7545 0.00% 0.00% TG 55:2 918.8484 21.75% 0.00% TG 56:8 920.770122.38% 0.00% TG 55:1 920.8640 21.76% 0.00% TG 56:7 922.7858 22.39% 0.00%TG 55:0; TG 56:7 922.8797 21.79% 0.00% TG 56:6 924.8015 21.80% 0.00% TG56:5 926.8171 22.42% 0.00% TG 56:4 928.8328 22.44% 0.00% TG 56:3930.8484 22.45% 0.00% TG 56:2 932.8641 22.47% 0.00% TG 56:1 934.879722.48% 0.00% TG 57:7 936.8014 0.00% 0.00% TG 56:0 936.8954 22.50% 0.00%TG 57:6 938.8171 22.51% 0.00% TG 58:12 940.7388 0.00% 0.00% TG 57:5940.8328 23.15% 0.00% TG 58:11 942.7545 23.79% 0.00% TG 57:4 942.848423.16% 0.00% TG 58:10 944.7701 23.81% 0.00% TG 57:3 (IS) 944.8640 23.18%0.00% TG 58:9 946.7858 23.82% 0.00% TG 57:2 946.8797 23.20% 0.00% TG58:8 948.8014 23.84% 0.00% TG 57:1; TG 58:8 948.8954 23.21% 0.00% TG58:7 950.8171 23.86% 0.00% TG 57:0; TG 58:7 950.9110 23.23% 0.00% TG58:6 952.8328 23.24% 0.00% TG 58:5 954.8484 23.89% 0.00% TG 58:4956.8641 23.90% 0.00% TG 58:3 958.8797 23.92% 0.00% TG 58:2 960.895423.93% 0.00% TG 59:8 962.8171 0.00% 0.00% TG 58:1 962.9110 23.95% 0.00%TG 59:7 964.8327 24.60% 0.00% TG 58:0 964.9267 23.96% 0.00% TG 60:13966.7545 0.00% 0.00% TG 59:6 966.8484 23.98% 0.00% TG 60:12 968.770125.29% 0.00% TG 59:5 968.8641 24.64% 0.00% TG 60:11 970.7858 25.30%0.00% TG 59:4 970.8797 24.65% 0.00% TG 60:10 972.8014 25.32% 0.00% TG59:3 972.8953 24.67% 0.00% TG 60:9 974.8171 25.33% 0.00% TG 59:2974.9110 24.68% 0.00% TG 60:8 976.8327 25.35% 0.00% TG 59:1 976.926624.70% 0.00% TG 59:1; TG 60:8 976.9267 24.70% 0.00% TG 60:7 978.848425.37% 0.00% TG 59:0; TG 60:7 978.9423 24.72% 0.00% TG 60:6 980.864124.73% 0.00% TG 60:5 982.8797 25.40% 0.00% TG 60:4 984.8954 25.41% 0.00%TG 60:3 986.9110 25.43% 0.00% TG 60:2 988.9267 25.45% 0.00% TG 60:1990.9423 25.46% 0.00% TG 60:0 992.9580 25.48% 0.00% TG 62:12 996.80140.00% 0.00% TG 62:11 998.8171 26.86% 0.00% TG 61:4 998.9110 0.00% 0.00%TG 62:10 1000.8327 26.88% 0.00% TG 61:3 1000.9266 26.20% 0.00% TG 62:91002.8484 26.89% 0.00% TG 61:2 1002.9423 26.22% 0.00% TG 62:8 1004.864026.91% 0.00% TG 62:7 1006.8797 26.92% 0.00% TG 62:6 1008.8953 26.94%0.00% TG 62:5 1010.9110 26.96% 0.00% TG 62:4 1012.9266 26.97% 0.00% TG62:3 1014.9423 26.99% 0.00% TG 62:2 1016.9579 27.01% 0.00% TG 62:11018.9736 27.02% 0.00% TG 64:12 1024.8327 0.00% 0.00% TG 64:11 1026.848428.46% 0.00% TG 63:4 1026.9423 0.00% 0.00% TG 64:10 1028.8640 28.48%0.00% TG 63:3 1028.9579 27.79% 0.00% TG 64:9 1030.8797 28.50% 0.00% TG64:8 1032.8953 28.52% 0.00% TG 64:7 1034.9110 28.53% 0.00% TG 64:61036.9266 28.55% 0.00% TG 64:5 1038.9423 28.57% 0.00% TG 64:4 1040.957928.58% 0.00% TG 66:12 1052.8640 0.00% 0.00% TG 66:11 1054.8797 30.12%0.00% TG 66:10 1056.8953 30.14% 0.00% TG 66:9 1058.9110 30.15% 0.00% TG66:8 1060.9266 30.17% 0.00% TG 66:7 1062.9423 30.19% 0.00% TG 66:61064.9579 30.21% 0.00% MG 10:0 264.2169 0.00% 0.00% MG 12:0 292.24820.00% 0.00% MG 14:1 318.2639 0.00% 0.00% MG 14:0 320.2795 2.59% 0.00% MG15:1 332.2795 0.00% 0.00% MG 15:0 334.2952 2.80% 0.00% MG 16:1 346.29520.00% 0.00% MG 16:0 348.3108 3.03% 0.00% MG 16:0 353.2662 0.00% 0.00% MG17:1 360.3108 0.00% 0.00% MG 17:0 362.3265 3.27% 0.00% MG 17:0 367.28190.00% 0.00% MG 18:4 368.2795 0.00% 0.00% Chol 369.3516 0.00% 0.00% CholD7 (IS) 376.3955 0.00% 0.00% Chol 404.3887 0.00% 0.00% Chol D7 (IS)411.4326 0.00% 0.00% MG 18:3 370.2952 3.50% 0.00% MG 18:2 372.3108 3.51%0.00% MG 18:1 374.3265 3.51% 0.00% MG 18:0 376.3421 3.52% 0.00% MG 18:1379.2819 0.00% 0.00% MG 18:0 381.2975 3.42% 0.00% MG 19:1 (IS) 388.34210.00% 0.00% MG 19:0 390.3578 3.78% 0.00% MG 19:1 (IS) 393.2975 0.00%0.00% MG 20:5 394.2952 0.00% 0.00% MG 20:4 396.3108 4.03% 0.00% MG 20:3398.3265 4.04% 0.00% MG 20:2 400.3421 4.04% 0.00% MG 20:1 402.3578 4.05%0.00% MG 21:1 416.3734 0.00% 0.00% MG 21:0 418.3891 4.34% 0.00% MG 22:6420.3108 4.35% 0.00% MG 22:5 422.3265 4.61% 0.00% MG 22:4 424.3421 4.61%0.00% MG 22:3 426.3587 4.62% 0.00% MG 22:2 428.3734 4.63% 0.00% MG 22:1430.3891 4.63% 0.00% MG 22:0 432.4047 4.64% 0.00% DG 24:2 (IS) 435.34690.00% 0.00% MG 23:1 444.4047 0.00% 0.00% MG 23:0 446.4204 4.95% 0.00% MG24:1 458.4204 0.00% 0.00% MG 24:0 460.4360 5.27% 0.00% DG 24:2 (IS)470.3840 0.00% 0.00% DG 28:0 530.4779 0.00% 0.00% DG 29:0 544.4935 0.00%0.00% DG 30:2 554.4779 0.00% 0.00% DG 30:1 556.4935 7.67% 0.00% DG 30:0558.5092 7.68% 0.00% DG 31:2 568.4935 0.00% 0.00% DG 31:1 570.5092 8.08%0.00% DG 31:0 572.5248 8.09% 0.00% DG 32:3 580.4936 0.00% 0.00% DG 32:2582.5092 8.50% 0.00% DG 32:1 584.5249 8.50% 0.00% DG 32:0 586.5405 8.51%0.00% DG 33:3 594.5092 0.00% 0.00% DG 33:2 596.5248 8.93% 0.00% DG 33:1598.5405 8.94% 0.00% DG 33:0 600.5561 8.95% 0.00% DG 34:4 606.5092 0.00%0.00% DG 34:3 608.5249 9.36% 0.00% DG 34:2 610.5405 9.37% 0.00% DG 34:1612.5562 9.38% 0.00% DG 34:0 614.5718 9.39% 0.00% DG 35:4 620.5248 0.00%0.00% DG 35:3 622.5405 9.82% 0.00% DG 35:2 624.5561 9.83% 0.00% DG 35:1626.5718 9.84% 0.00% DG 35:0 628.5875 9.85% 0.00% DG 36:5 632.5249 0.00%0.00% DG 36:4 634.5405 10.28% 0.00% DG 36:3 636.5561 10.29% 0.00% DG36:2 638.5718 10.30% 0.00% DG 36:1 640.5874 10.31% 0.00% DG 37:7642.5092 0.00% 0.00% DG 36:0 642.6031 10.32% 0.00% DG 37:5 646.54050.00% 0.00% DG 37:4 648.5561 0.00% 0.00% DG 37:3 650.5718 10.77% 0.00%DG 37:2 652.5874 10.78% 0.00% DG 37:1 654.6031 10.79% 0.00% DG 38:7656.5248 0.00% 0.00% DG 37:0 656.6188 10.80% 0.00% DG 38:6 658.540511.23% 0.00% DG 38:5 660.5561 11.24% 0.00% DG 38:4 662.5718 11.26% 0.00%DG 38:3 664.5874 11.27% 0.00% DG 38:2 666.6031 11.28% 0.00% DG 38:1668.6187 11.29% 0.00% DG 38:0 670.6344 11.30% 0.00% DG 39:6 672.556211.31% 0.00% DG 39:5 674.5718 11.75% 0.00% DG 39:4 676.5875 11.76% 0.00%DG 40:10; DG 39:3 678.5092 11.77% 0.00% DG 40:9; DG 39:2 680.524912.223% 0.000% DG 40:8; DG 39:1 682.5405 12.23% 0.00% DG 40:7; DG 39:0684.5562 12.25% 0.00% DG 40:6 686.5718 12.26% 0.00% DG 40:5 688.587412.27% 0.00% DG 40:4 690.6031 12.28% 0.00% DG 40:3 692.6187 12.29% 0.00%DG 40:2 694.6344 12.30% 0.00% DG 40:1 696.6500 12.31% 0.00% DG 40:0698.6657 12.32% 0.00% DG 41:6 700.5675 12.33% 0.00% DG 41:5 702.360312.80% 0.00% DG 41:4 704.6188 12.81% 0.00% DG 42:10; DG 41:3 706.540512.82% 0.00% DG 42:9; DG 41:2 708.5562 13.29% 0.00% DG 42:8; DG 41:1710.5718 13.30% 0.00% DG 42:7; DG 41:0 712.5875 13.32% 0.00% DG 42:6714.6031 13.33% 0.00% DG 42:5 716.6188 13.34% 0.00% DG 42:4 718.634413.35% 0.00% DG 42:3 720.6501 13.36% 0.00% DG 42:2 722.6657 13.37% 0.00%DG 42:1 724.6814 13.39% 0.00% DG 42:0 726.6970 13.40% 0.00% DG 44:0754.7283 0.00% 0.00% Coenzyme Q10 880.7177 0.00% 0.00% Cer d30:1 (IS)464.4462 0.00% 0.00% Cer d18:1/12:2 478.4255 0.00% 0.00% Cer d18:1/12:1480.4411 6.08% 0.00% Cer d18:0/12:2 480.4412 0.00% 0.00% Cer d18:1/12:0(IS) 482.4568 6.08% 0.00% Cer d18:0/12:1 (IS) 482.4568 6.08% 0.00% Cerd18:0/12:0 484.4725 6.09% 0.00% Cer d18:1/13:2 492.4411 0.00% 0.00% Cerd32:1 492.4775 0.00% 0.00% Cer d18:1/13:1; Cer d18:1/12:2 (1OH) 494.45686.45% 0.00% Cer d18:0/13:2 494.4568 0.00% 0.00% Cer d18:1/13:0; Cerd18:1/12:1 (1OH) 496.4724 6.46% 0.00% Cer d18:0/13:1; Cer d18:0/12:2(1OH) 496.4725 6.46% 0.00% Cer d18:1/12:0 (1OH) 498.4517 6.46% 0.00% Cerd18:0/13:0; Cer d18:0/12:1 (1OH) 498.4881 6.46% 0.00% Cer d18:0/12:0(1OH) 500.4674 6.47% 0.00% Cer d18:1/14:2 506.4568 0.00% 0.00% Cerd18:1/14:1; Cer d18:1/13:2 (1OH) 508.4724 6.83% 0.00% Cer d18:0/14:2508.4725 0.00% 0.00% Cer d18:1/14:0; Cer d18:1/13:0 (1OH) 510.4808 6.84%0.00% Cer d18:0/14:1; Cer d18:0/13:2 (1OH) 510.4881 6.84% 0.00% Cerd18:1/13:0 (1OH) 512.4674 6.85% 0.00% Cer d18:0/14:0; Cer d18:0/13:0(1OH) 512.4965 6.85% 0.00% Cer d18:0/13:0 (1OH) 514.4831 6.86% 0.00% Cerd34:2 518.4932 0.00% 0.00% Cer d18:1/15:2 520.4724 0.00% 0.00% Cer d34:1520.5088 7.41% 0.00% Cer d18:0/15:2 522.4881 0.00% 0.00% Cer d18:1/15:1;Cer d18:1/14:2 (1OH) 522.4881 7.23% 0.00% Cer d34:0 522.5245 7.42% 0.00%Cer d18:1/15:0; Cer d18:1/14:1 (1OH) 524.5037 7.24% 0.00% Cerd18:0/15:1; Cer d18:0/14:2 (1OH) 524.5038 7.24% 0.00% Cer d18:1/14:0(1OH) 526.4830 7.24% 0.00% Cer d18:0/15:0; Cer d18:0/14:1 (1OH) 526.51947.24% 0.00% Cer d18:0/14:0 (1OH) 528.4987 7.25% 0.00% Cer d18:1/16:2534.4881 0.00% 0.00% Cer d35:1 534.5245 0.00% 0.00% Cer d18:1/16:1; Cerd18:1/15:2 (1OH) 536.5037 7.63% 0.00% Cer d18:0/16:2 536.5038 0.00%0.00% Cer d18:0/16:1; Cer d18:0/15:2 (1OH) 538.5194 7.64% 0.00% Cerd18:1/16:0; Cer d18:1/15:1 (1OH) 538.5194 7.64% 0.00% Cer d18:1/15:0(1OH) 540.4987 7.65% 0.00% Cer d18:0/16:0; Cer d18:0/15:1 (1OH) 540.53517.65% 0.00% Cer d18:0/15:0 (1OH) 542.5144 7.66% 0.00% Cer d36:2 546.52450.00% 0.00% Cer d18:1/17:2 548.5037 0.00% 0.00% Cer d36:1 548.5401 8.27%0.00% Cer d18:0/17:2 550.5194 0.00% 0.00% Cer d18:1/17:1; Cer d18:1/16:2(1OH) 550.5194 8.05% 0.00% Cer d36:0 550.5558 8.28% 0.00% Cerd18:1/17:0; Cer d18:1/16:1 (1OH) 552.5350 8.06% 0.00% Cer d18:0/17:1;Cer d18:0/16:2 (1OH) 552.5351 8.06% 0.00% Cer d18:1/16:0 (1OH) 554.51438.07% 0.00% Cer d18:0/17:0; Cer d18:0/16:1 (1OH) 554.5507 8.07% 0.00%Cer d18:0/16:0 (1OH) 556.5300 8.08% 0.00% Cer d18:1/18:3 560.5037 0.00%0.00% Cer d18:1/18:2 562.5193 8.48% 0.00% Cer d37:1 562.5558 0.00% 0.00%Cer d18:0/18:2 564.5350 0.00% 0.00% Cer d18:1/18:1; Cer d18:1/17:2 (1OH)564.5350 8.49% 0.00% Cer d18:1/18:0; Cer d18:1/17:1 (1OH) 566.5506 8.50%0.00% Cer d18:0/18:1; Cer d18:0/17:2 (1OH) 566.5507 8.50% 0.00% Cerd18:1/17:0 (1OH) 568.5300 8.51% 0.00% Cer d18:0/18:0; Cer d18:0/17:1(1OH) 568.5663 8.51% 0.00% Cer d18:0/17:0 (1OH) 570.5457 8.51% 0.00% Cerd38:2 574.5558 0.00% 0.00% Cer d18:1/19:2 576.5349 0.00% 0.00% Cer d38:1576.5714 9.17% 0.00% Cer d18:0/19:2 578.5506 0.00% 0.00% Cer d18:1/19:1;Cer d18:1/18:2 (1OH) 578.5506 8.93% 0.00% Cer d38:0 578.5871 9.18% 0.00%Cer d18:1/19:0; Cer d18:1/18:1 (1OH) 580.5662 8.94% 0.00% Cerd18:0/19:1; Cer d18:0/18:2 (1OH) 580.5663 8.94% 0.00% Cer d18:1/18:0(1OH) 582.5383 8.95% 0.00% Cer d18:0/19:0; Cer d18:0/18:1 (1OH) 582.58198.95% 0.00% Cer d18:0/18:0 (1OH) 584.5540 8.96% 0.00% Cer d18:1/20:2590.5506 0.00% 0.00% Cer d39:1 590.5871 0.00% 0.00% Cer d18:1/20:1; Cerd18:1/19:2 (1OH) 592.5662 9.39% 0.00% Cer d18:0/20:2 592.5663 0.00%0.00% Cer d18:0/20:1; Cer d18:0/19:2 (1OH) 594.5819 9.40% 0.00% Cerd18:1/20:0; Cer d18:1/19:1 (1OH) 594.5819 9.40% 0.00% Cer d18:1/19:0(1OH) 596.5613 9.41% 0.00% Cer d18:0/20:0; Cer d18:0/19:1 (1OH) 596.59769.41% 0.00% Cer d18:0/19:0 (1OH) 598.5770 9.42% 0.00% Cer d40:2 602.58710.00% 0.00% Cer d18:1/21:2 604.5662 0.00% 0.00% Cer d40:1 604.602710.11% 0.00% Cer d18:0/21:2 606.5819 0.00% 0.00% Cer d18:1/21:1; Cerd18:1/20:2 (1OH) 606.5819 9.86% 0.00% Cer d40:0 606.6184 10.13% 0.00%Cer d18:1/21:0; Cer d18:1/20:1 (1OH) 608.5975 9.87% 0.00% Cerd18:0/21:1; Cer d18:0/20:2 (1OH) 608.5976 9.87% 0.00% Cer d18:1/20:0(OH) 610.5769 9.88% 0.00% Cer d18:0/21:0; Cer d18:0/20:1 (1OH) 610.61329.88% 0.00% Cer d18:0/20:0 (1OH) 612.5926 9.89% 0.00% Cer d18:1/22:2618.5818 0.00% 0.00% Cer d41:1 618.6184 0.00% 0.00% Cer d18:0/22:2620.5975 0.00% 0.00% Cer d18:1/22:1; Cer d18:1/21:2 (1OH) 620.597510.34% 0.00% Cer d41:0 620.6340 10.62% 0.00% Cer d18:1/22:0; Cerd18:1/21:1 (1OH) 622.6131 10.35% 0.00% Cer d18:0/22:1; Cer d18:0/21:2(1OH) 622.6132 10.35% 0.00% Cer d18:1/21:0 (1OH) 624.5926 10.36% 0.00%Cer d18:0/22:0; Cer d18:0/21:1 (1OH) 624.6288 10.36% 0.00% Cerd18:0/21:0 (1OH) 626.6083 10.37% 0.00% Cer d42:3 628.6027 0.00% 0.00%Cer d42:2 630.6184 11.10% 0.00% Cer d18:1/23:2 632.5974 0.00% 0.00% Cerd42:1 632.6340 11.11% 0.00% Cer d18:0/23:2 634.6131 0.00% 0.00% Cerd18:1/23:1; Cer d18:1/22:2 (1OH) 634.6131 10.83% 0.00% Cer d42:0634.6497 11.12% 0.00% Cer d18:1/23:0; Cer d18:1/22:1 (1OH) 636.628710.84% 0.00% Cer d18:0/23:1; Cer d18:0/22:2 (1OH) 636.6288 10.84% 0.00%Cer d18:1/22:0 (1OH) 638.6082 10.85% 0.00% Cer d18:0/23:0; Cerd18:0/22:1 (1OH) 638.6444 10.85% 0.00% HexCer d18:1/12:2 640.4784 10.86%0.00% Cer d18:0/22:0 (1OH) 640.6239 10.86% 0.00% HexCer d18:1/12:1642.4940 9.58% 0.00% HexCer d18:0/12:2 642.4941 10.87% 0.00% HexCerd18:1/12:0 (IS) 644.5097 9.59% 0.00% HexCer d18:0/12:1 (IS) 644.50979.59% 0.00% Cer d43:2 644.6340 0.00% 0.00% HexCer d18:0/12:0 646.52549.60% 0.00% Cer d18:1/24:2 646.6131 9.60% 0.00% Cer d43:1 646.649711.63% 0.00% Cer d18:1/24:1; Cer d18:1/23:2 (1OH) 648.6287 11.34% 0.00%Cer d18:0/24:2 648.6288 9.61% 0.00% Cer d43:0 648.6653 11.64% 0.00% Cerd18:0/24:1; Cer d18:0/23:2 (1OH) 650.6444 11.35% 0.00% Cer d18:1/24:0;Cer d18:1/23:1 (1OH) 650.6444 11.35% 0.00% Cer d18:1/23:0 (1OH) 652.623911.36% 0.00% Cer d18:0/24:0; Cer d18:0/23:1 (1OH) 652.6601 11.36% 0.00%HexCer d18:1/13:2 654.4940 11.07% 0.00% Cer d18:0/23:0 (1OH) 654.639611.07% 0.00% HexCer d18:1/13:1; HexCer d18:1/12:2 (1OH) 656.5097 10.03%0.00% HexCer d18:0/13:2 656.5097 11.08% 0.00% Cer d44:3 656.6340 0.00%0.00% HexCer d18:1/13:0; HexCer d18:1/12:1 (1OH) 658.5253 10.04% 0.00%HexCer d18:0/13:1; HexCer d18:0/12:2 (1OH) 658.5254 10.04% 0.00% Cerd44:2 658.6497 12.15% 0.00% HexCer d18:1/12:0 (1OH); Cer d18:1/25:2660.5046 10.05% 0.00% HexCer d18:0/13:0; HexCer d18:0/12:1 (1OH)660.5410 10.05% 0.00% Cer d44:1 660.6653 12.16% 0.00% HexCer d18:0/12:0(1OH); Cer d18:0/25:2 662.5203 10.06% 0.00% Cer d18:1/25:1; Cerd18:1/24:2 (1OH) 662.6444 10.06% 0.00% Cer d44:0 662.6810 12.17% 0.00%Cer d18:1/25:0; Cer d18:1/24:1 (1OH) 664.6600 11.86% 0.00% Cerd18:0/25:1; Cer d18:0/24:2 (1OH) 664.6601 11.86% 0.00% Cer d18:1/24:0(1OH) 666.6395 11.88% 0.00% Cer d18:0/25:0; Cer d18:0/24:1 (1OH)666.6757 11.88% 0.00% HexCer d18:1/14:2 668.5097 11.58% 0.00% Cerd18:0/24:0 (1OH) 668.6552 11.89% 0.00% HexCer d18:1/14:1; HexCerd18:1/13:2 (1OH) 670.5253 10.49% 0.00% HexCer d18:0/14:2 670.5254 11.59%0.00% HexCer d18:1/14:0; HexCer d18:1/13:0 (1OH) 672.5337 10.50% 0.00%HexCer d18:0/14:1; HexCer d18:0/13:2 (1OH) 672.5410 10.50% 0.00% HexCerd18:0/14:0; HexCer d18:0/13:0 (1OH) 674.5494 10.51% 0.00% Cerd18:1/26:2; HexCer d18:1/13:0 (1OH) 674.6443 10.51% 0.00% Cerd18:0/26:2; HexCer d18:0/13:0 (1OH) 676.6600 10.52% 0.00% Cerd18:1/26:1; Cer d18:1/25:2 (1OH) 676.6600 12.38% 0.00% Cer d18:1/26:0;Cer d18:1/25:1 (1OH) 678.6756 12.39% 0.00% Cer d18:0/26:1; Cerd18:0/25:2 (1OH) 678.6757 12.39% 0.00% Cer d18:1/25:0 (1OH) 680.655212.41% 0.00% Cer d18:0/26:0; Cer d18:0/25:1 (1OH) 680.6913 12.41% 0.00%HexCer d18:1/15:2 682.5253 12.11% 0.00% Cer d18:0/25:0 (1OH) 682.670912.42% 0.00% HexCer d18:0/15:2 684.5410 12.11% 0.00% HexCer d18:1/15:1;HexCer d18:1/14:2 (1OH) 684.5410 10.96% 0.00% HexCer d18:1/15:0; HexCerd18:1/14:1 (1OH) 686.5566 10.97% 0.00% HexCer d18:0/15:1; HexCerd18:0/14:2 (1OH) 686.5567 10.97% 0.00% HexCer d18:1/14:0 (1OH); Cerd18:1/27:2 688.5359 10.98% 0.00% HexCer d18:0/15:0; HexCer d18:0/14:1(1OH) 688.5723 10.98% 0.00% HexCer d18:0/14:0 (1OH); Cer d18:0/27:2690.5516 10.99% 0.00% Cer d18:1/27:1; Cer d18:1/26:2 (1OH) 690.675610.90% 0.00% Cer d18:1/27:0; Cer d18:1/26:1 (1OH) 692.6912 12.94% 0.00%Cer d18:0/27:1; Cer d18:0/26:2 (1OH) 692.6913 11.00% 0.00% Cerd18:1/26:0 (1OH) 694.6708 12.95% 0.00% Cer d18:0/27:0; Cer d18:0/26:1(1OH) 694.7069 12.95% 0.00% HexCer d18:1/16:2 696.5410 12.63% 0.00% Cerd18:0/26:0 (1OH) 696.6865 12.96% 0.00% HexCer d18:1/16:1; HexCerd18:1/15:2 (1OH) 698.5566 11.44% 0.00% HexCer d18:0/16:2 698.5567 12.64%0.00% HexCer d18:0/16:1; HexCer d18:0/15:2 (1OH) 700.5723 11.45% 0.00%HexCer d18:1/16:0; HexCer d18:1/15:1 (1OH) 700.5723 11.45% 0.00% HexCerd18:0/16:0; HexCer d18:0/15:1 (1OH) 702.5880 11.46% 0.00% Cerd18:1/28:2; HexCer d18:1/15:0 (1OH) 702.6756 11.46% 0.00% Cerd18:1/28:1; Cer d18:1/27:2 (1OH) 704.6912 13.48% 0.00% Cer d18:0/28:2;HexCer d18:0/15:0 (1OH) 704.6913 11.47% 0.00% Cer d18:0/28:1; Cerd18:0/27:2 (1OH) 706.7069 13.49% 0.00% Cer d18:1/28:0; Cer d18:1/27:1(1OH) 706.7069 13.49% 0.00% Cer d18:1/27:0 (1OH) 708.6865 13.50% 0.00%Cer d18:0/28:0; Cer d18:0/27:1 (1OH) 708.7226 13.50% 0.00% HexCerd18:1/17:2 710.5566 13.17% 0.00% Cer d18:0/27:0 (1OH) 710.7022 13.51%0.00% HexCer d18:0/17:2 712.5723 13.18% 0.00% HexCer d18:1/17:1; HexCerd18:1/16:2 (1OH) 712.5723 11.93% 0.00% HexCer d18:1/17:0; HexCerd18:1/16:1 (1OH) 714.5879 11.95% 0.00% HexCer d18:0/17:1; HexCerd18:0/16:2 (1OH) 714.5880 11.95% 0.00% HexCer d18:1/16:0 (1OH) 716.567211.96% 0.00% HexCer d18:0/17:0; HexCer d18:0/16:1 (1OH) 716.6036 11.96%0.00% HexCer d18:0/16:0 (1OH) 718.5829 11.97% 0.00% Cer d18:1/29:1; Cerd18:1/28:2 (1OH) 718.7069 11.97% 0.00% Cer d18:1/29:0; Cer d18:1/28:1(1OH) 720.7225 14.06% 0.00% Cer d18:0/29:1; Cer d18:0/28:2 (1OH)720.7226 11.98% 0.00% Cer d18:1/28:0 (1OH) 722.7201 14.07% 0.00% Cerd18:0/29:0; Cer d18:0/28:1 (1OH) 722.7382 14.07% 0.00% HexCer d18:1/18:2724.5722 13.73% 0.00% Cer d18:0/28:0 (1OH) 724.7358 14.08% 0.00% HexCerd18:0/18:2 726.5879 13.74% 0.00% HexCer d18:1/18:1; HexCer d18:1/17:2(1OH) 726.5879 12.44% 0.00% HexCer d18:1/18:0; HexCer d18:1/17:1 (1OH)728.6035 12.45% 0.00% HexCer d18:0/18:1; HexCer d18:0/17:2 (1OH)728.6036 12.45% 0.00% HexCer d18:1/17:0 (1OH) 730.5829 12.46% 0.00%HexCer d18:0/18:0; HexCer d18:0/17:1 (1OH) 730.6192 12.46% 0.00% HexCerd18:0/17:0 (1OH) 732.5986 12.47% 0.00% Cer d18:1/30:1; Cer d18:1/29:2(1OH) 732.7228 12.47% 0.00% Cer d18:1/30:0; Cer d18:1/29:1 (1OH)734.7381 14.64% 0.00% Cer d18:0/30:1; Cer d18:0/29:2 (1OH) 734.738512.49% 0.00% Cer d18:1/29:0 (1OH) 736.7178 14.65% 0.00% Cer d18:0/30:0;Cer d18:0/29:1 (1OH) 736.7538 14.65% 0.00% HexCer d18:1/19:2 738.587814.29% 0.00% Cer d18:0/29:0 (1OH) 738.7335 14.66% 0.00% HexCerd18:0/19:2 740.6035 14.31% 0.00% HexCer d18:1/19:1; HexCer d18:1/18:2(1OH) 740.6035 12.96% 0.00% HexCer d18:1/19:0; HexCer d18:1/18:1 (1OH)742.6191 12.97% 0.00% HexCer d18:0/19:1; HexCer d18:0/18:2 (1OH)742.6192 12.97% 0.00% HexCer d18:1/18:0 (1OH) 744.5912 12.98% 0.00%HexCer d18:0/19:0; HexCer d18:0/18:1 (1OH) 744.6348 12.98% 0.00% HexCerd18:0/18:0 (1OH) 746.6069 12.99% 0.00% HexCer d18:1/20:2 752.6035 12.99%0.00% HexCer d18:1/20:1; HexCer d18:1/19:2 (1OH) 754.6191 13.49% 0.00%HexCer d18:0/20:2 754.6192 13.01% 0.00% HexCer d18:0/20:1; HexCerd18:0/19:2 (1OH) 756.6348 13.50% 0.00% HexCer d18:1/20:0; HexCerd18:1/19:1 (1OH) 756.6348 13.50% 0.00% HexCer d18:1/19:0 (1OH) 758.614213.52% 0.00% HexCer d18:0/20:0; HexCer d18:0/19:1 (1OH) 758.6505 13.52%0.00% HexCer d18:0/19:0 (1OH) 760.6299 13.53% 0.00% HexCer d18:1/21:2766.6191 0.00% 0.00% HexCer d18:0/21:2 768.6348 0.00% 0.00% HexCerd18:1/21:1; HexCer d18:1/20:2 (1OH) 768.6348 14.04% 0.00% HexCerd18:1/21:0; HexCer d18:1/20:1 (1OH) 770.6504 14.05% 0.00% HexCerd18:0/21:1; HexCer d18:0/20:2 (1OH) 770.6505 14.05% 0.00% HexCerd18:1/20:0 (OH) 772.6298 14.06% 0.00% HexCer d18:0/21:0; HexCerd18:0/20:1 (1OH) 772.6661 14.06% 0.00% HexCer d18:0/20:0 (OH) 774.645514.07% 0.00% HexCer d18:1/22:2 780.6347 0.00% 0.00% HexCer d18:0/22:2782.6504 0.00% 0.00% HexCer d18:1/22:1; HexCer d18:1/21:2 (1OH) 782.650414.59% 0.00% HexCer d18:1/22:0; HexCer d18:1/21:1 (1OH) 784.6660 14.60%0.00% HexCer d18:0/22:1; HexCer d18:0/21:2 (1OH) 784.6661 14.60% 0.00%HexCer d18:1/21:0 (1OH) 786.6455 14.62% 0.00% HexCer d18:0/22:0; HexCerd18:0/21:1 (1OH) 786.6817 14.62% 0.00% HexCer d18:0/21:0 (1OH) 788.661214.63% 0.00% HexCer d18:1/23:2 794.6503 0.00% 0.00% HexCer d18:0/23:2796.6660 0.00% 0.00% HexCer d18:1/23:1; HexCer d18:1/22:2 (1OH) 796.666015.16% 0.00% HexCer d18:1/23:0; HexCer d18:1/22:1 (1OH) 798.6816 15.17%0.00% HexCer d18:0/23:1; HexCer d18:0/22:2 (1OH) 798.6817 15.17% 0.00%HexCer d18:1/22:0 (1OH) 800.6611 15.18% 0.00% HexCer d18:0/23:0; HexCerd18:0/22:1 (1OH) 800.6973 15.18% 0.00% Hex2Cer d18:1/12:2 802.531414.84% 0.00% HexCer d18:0/22:0 (1OH) 802.6768 15.20% 0.00% Hex2Cerd18:1/12:1 804.5470 13.55% 0.00% Hex2Cer d18:0/12:2 804.5471 14.85%0.00% Hex2Cer d18:0/12:1 (IS) 806.5627 13.56% 0.00% Hex2Cer d18:1/12:0(IS) 806.5627 13.56% 0.00% Hex2Cer d18:0/12:0 808.5784 13.57% 0.00%HexCer d18:1/24:2 808.6660 13.57% 0.00% HexCer d18:1/24:1; HexCerd18:1/23:2 (1OH) 810.6816 15.74% 0.00% HexCer d18:0/24:2 810.6817 13.58%0.00% HexCer d18:0/24:1; HexCer d18:0/23:2 (1OH) 812.6973 15.75% 0.00%HexCer d18:1/24:0; HexCer d18:1/23:1 (1OH) 812.6973 15.75% 0.00% HexCerd18:1/23:0 (1OH) 814.6768 15.77% 0.00% HexCer d18:0/24:0; HexCerd18:0/23:1 (1OH) 814.7130 15.77% 0.00% Hex2Cer d18:1/13:2 816.547015.41% 0.00% HexCer d18:0/23:0 (1OH) 816.6925 15.78% 0.00% Hex2Cerd18:0/13:2 818.5627 15.42% 0.00% Hex2Cer d18:1/13:1; Hex2Cer d18:1/12:2(1OH) 818.5627 14.07% 0.00% Hex2Cer d18:1/13:0; Hex2Cer d18:1/12:1 (1OH)820.5783 14.08% 0.00% Hex2Cer d18:0/13:1; Hex2Cer d18:0/12:2 (1OH)820.5784 14.08% 0.00% Hex2Cer d18:1/12:0 (1OH); HexCer d18:1/25:2822.5576 14.09% 0.00% Hex2Cer d18:0/13:0; Hex2Cer d18:0/12:1 (1OH)822.5940 14.09% 0.00% Hex2Cer d18:0/12:0 (1OH); HexCer d18:0/25:2824.5733 14.10% 0.00% HexCer d18:1/25:1; HexCer d18:1/24:2 (1OH)824.6973 13.79% 0.00% HexCer d18:1/25:0; HexCer d18:1/24:1 (1OH)826.7129 16.35% 0.00% HexCer d18:0/25:1; HexCer d18:0/24:2 (1OH)826.7130 13.80% 0.00% HexCer d18:1/24:0 (1OH) 828.6924 16.36% 0.00%HexCer d18:0/25:0; HexCer d18:0/24:1 (1OH) 828.7286 16.36% 0.00% Hex2Cerd18:1/14:2 830.5627 15.99% 0.00% HexCer d18:0/24:0 (1OH) 830.7081 16.37%0.00% Hex2Cer d18:1/14:1; Hex2Cer d18:1/13:2 (1OH) 832.5783 14.60% 0.00%Hex2Cer d18:0/14:2 832.5784 16.00% 0.00% Hex2Cer d18:1/14:0; Hex2Cerd18:1/13:1 (1OH) 834.5867 14.61% 0.00% Hex2Cer d18:0/14:1; Hex2Cerd18:0/13:2 (1OH) 834.5940 14.61% 0.00% Hex2Cer d18:0/14:0; Hex2Cerd18:0/13:1 (1OH) 836.6024 14.63% 0.00% HexCer d18:1/26:2; Hex2Cerd18:1/13:0 (1OH) 836.6972 14.63% 0.00% HexCer d18:0/26:2; Hex2Cerd18:0/13:0 (1OH) 838.7129 14.64% 0.00% HexCer d18:1/26:1; HexCerd18:1/25:2 (1OH) 838.7129 16.94% 0.00% HexCer d18:1/26:0; HexCerd18:1/25:1 (1OH) 840.7285 16.95% 0.00% HexCer d18:0/26:1; HexCerd18:0/25:2 (1OH) 840.7286 16.95% 0.00% HexCer d18:1/25:0 (1OH) 842.708116.96% 0.00% HexCer d18:0/26:0; HexCer d18:0/25:1 (1OH) 842.7442 16.96%0.00% Hex2Cer d18:1/15:2 844.5783 16.59% 0.00% HexCer d18:0/25:0 (1OH)844.7238 16.98% 0.00% Hex2Cer d18:0/15:2 846.5940 16.60% 0.00% Hex2Cerd18:1/15:1; Hex2Cer d18:1/14:2 (1OH) 846.5940 15.15% 0.00% Hex2Cerd18:1/15:0; Hex2Cer d18:1/14:1 (1OH) 848.6096 15.16% 0.00% Hex2Cerd18:0/15:1; Hex2Cer d18:0/14:2 (1OH) 848.6097 15.16% 0.00% Hex2Cerd18:1/14:0 (1OH); HexCer d18:1/27:2 850.5889 15.17% 0.00% Hex2Cerd18:0/15:0; Hex2Cer d18:0/14:1 (1OH) 850.6253 15.17% 0.00% Hex2Cerd18:0/14:0 (1OH); HexCer d18:0/27:2 852.6046 15.18% 0.00% HexCerd18:1/27:1; HexCer d18:1/26:2 (1OH) 852.7285 14.85% 0.00% HexCerd18:1/27:0; HexCer d18:1/26:1 (1OH) 854.7441 17.57% 0.00% HexCerd18:0/27:1; HexCer d18:0/26:2 (1OH) 854.7442 14.86% 0.00% HexCerd18:1/26:0 (1OH) 856.7237 17.58% 0.00% HexCer d18:0/27:0; HexCerd18:0/26:1 (1OH) 856.7598 17.58% 0.00% Hex2Cer d18:1/16:2 858.594017.19% 0.00% HexCer d18:0/26:0 (1OH) 858.7394 17.59% 0.00% Hex2Cerd18:1/16:1; Hex2Cer d18:1/15:2 (1OH) 860.6096 15.71% 0.00% Hex2Cerd18:0/16:2 860.6097 17.20% 0.00% Hex2Cer d18:0/16:1; Hex2Cer d18:0/15:2(1OH) 862.6253 15.72% 0.00% Hex2Cer d18:1/16:0; Hex2Cer d18:1/15:1 (1OH)862.6253 15.72% 0.00% Hex2Cer d18:0/16:0; Hex2Cer d18:0/15:1 (1OH)864.6410 15.73% 0.00% HexCer d18:1/28:2; Hex2Cer d18:1/15:0 (1OH)864.7285 15.73% 0.00% HexCer d18:1/28:1; HexCer d18:1/27:2 (1OH)866.7441 18.18% 0.00% HexCer d18:0/28:2; Hex2Cer d18:0/15:0 (1OH)866.7442 15.74% 0.00% HexCer d18:0/28:1; HexCer d18:0/27:2 (1OH)868.7598 18.20% 0.00% HexCer d18:1/28:0; HexCer d18:1/27:1 (1OH)868.7598 18.20% 0.00% HexCer d18:1/27:0 (1OH) 870.7394 18.21% 0.00%HexCer d18:0/28:0; HexCer d18:0/27:1 (1OH) 870.7755 18.21% 0.00% Hex2Cerd18:1/17:2 872.6096 17.81% 0.00% HexCer d18:0/27:0 (1OH) 872.7551 18.22%0.00% Hex2Cer d18:0/17:2 874.6253 17.82% 0.00% Hex2Cer d18:1/17:1;Hex2Cer d18:1/16:2 (1OH) 874.6253 16.28% 0.00% Hex2Cer d18:1/17:0;Hex2Cer d18:1/16:1 (1OH) 876.6409 16.29% 0.00% Hex2Cer d18:0/17:1;Hex2Cer d18:0/16:2 (1OH) 876.6410 16.29% 0.00% Hex2Cer d18:1/16:0 (1OH)878.6202 16.30% 0.00% Hex2Cer d18:0/17:0; Hex2Cer d18:0/16:1 (1OH)878.6566 16.30% 0.00% Hex2Cer d18:0/16:0 (1OH) 880.6359 16.31% 0.00%HexCer d18:1/29:1; HexCer d18:1/28:2 (1OH) 880.7598 15.96% 0.00% HexCerd18:1/29:0; HexCer d18:1/28:1 (1OH) 882.7754 18.84% 0.00% HexCerd18:0/29:1; HexCer d18:0/28:2 (1OH) 882.7755 15.97% 0.00% HexCerd18:1/28:0 (1OH) 884.7730 18.85% 0.00% HexCer d18:0/29:0; HexCerd18:0/28:1 (1OH) 884.7911 18.85% 0.00% Hex2Cer d18:1/18:2 886.625218.44% 0.00% HexCer d18:0/28:0 (1OH) 886.7887 18.87% 0.00% Hex2Cerd18:0/18:2 888.6409 18.45% 0.00% Hex2Cer d18:1/18:1; Hex2Cer d18:1/17:2(1OH) 888.6409 16.86% 0.00% Hex2Cer d18:1/18:0; Hex2Cer d18:1/17:1 (1OH)890.6565 16.87% 0.00% Hex2Cer d18:0/18:1; Hex2Cer d18:0/17:2 (1OH)890.6566 16.87% 0.00% Hex2Cer d18:1/17:0 (1OH) 892.6359 16.88% 0.00%Hex2Cer d18:0/18:0; Hex2Cer d18:0/17:1 (1OH) 892.6722 16.88% 0.00%Hex2Cer d18:0/17:0 (1OH) 894.6516 16.90% 0.00% HexCer d18:1/30:1; HexCerd18:1/29:2 (1OH) 894.7757 16.53% 0.00% HexCer d18:1/30:0; HexCerd18:1/29:1 (1OH) 896.7910 19.49% 0.00% HexCer d18:0/30:1; HexCerd18:0/29:2 (1OH) 896.7914 16.54% 0.00% HexCer d18:1/29:0 (1OH) 898.770719.51% 0.00% HexCer d18:0/30:0; HexCer d18:0/29:1 (1OH) 898.8067 19.51%0.00% Hex2Cer d18:1/19:2 900.6408 19.08% 0.00% HexCer d18:0/29:0 (1OH)900.7864 19.52% 0.00% Hex2Cer d18:0/19:2 902.6565 19.09% 0.00% Hex2Cerd18:1/19:1; Hex2Cer d18:1/18:2 (1OH) 902.6565 17.45% 0.00% Hex2Cerd18:1/19:0; Hex2Cer d18:1/18:1 (1OH) 904.6721 17.47% 0.00% Hex2Cerd18:0/19:1; Hex2Cer d18:0/18:2 (1OH) 904.6722 17.47% 0.00% Hex2Cerd18:1/18:0 (1OH) 906.6442 17.48% 0.00% Hex2Cer d18:0/19:0; Hex2Cerd18:0/18:1 (1OH) 906.6878 17.48% 0.00% Hex2Cer d18:0/18:0 (1OH) 908.659917.49% 0.00% Hex2Cer d18:1/20:2 914.6565 0.00% 0.00% Hex2Cer d18:1/20:1;Hex2Cer d18:1/19:2 (1OH) 916.6721 18.06% 0.00% Hex2Cer d18:0/20:2916.6722 0.00% 0.00% Hex2Cer d18:0/20:1; Hex2Cer d18:0/19:2 (1OH)918.6878 18.07% 0.00% Hex2Cer d18:1/20:0; Hex2Cer d18:1/19:1 (1OH)918.6878 18.07% 0.00% Hex2Cer d18:1/19:0 (1OH) 920.6672 18.09% 0.00%Hex2Cer d18:0/20:0; Hex2Cer d18:0/19:1 (1OH) 920.7035 18.09% 0.00%Hex2Cer d18:0/19:0 (1OH) 922.6829 18.10% 0.00% Hex2Cer d18:1/21:2928.6721 0.00% 0.00% Hex2Cer d18:0/21:2 930.6878 0.00% 0.00% Hex2Cerd18:1/21:1; Hex2Cer d18:1/20:2 (1OH) 930.6878 18.68% 0.00% Hex2Cerd18:1/21:0; Hex2Cer d18:1/20:1 (1OH) 932.7034 18.69% 0.00% Hex2Cerd18:0/21:1; Hex2Cer d18:0/20:2 (1OH) 932.7035 18.69% 0.00% Hex2Cerd18:1/20:0 (OH) 934.6828 18.70% 0.00% Hex2Cer d18:0/21:0; Hex2Cerd18:0/20:1 (1OH) 934.7191 18.70% 0.00% Hex2Cer d18:0/20:0 (OH) 936.698518.72% 0.00% Hex2Cer d18:1/22:2 942.6877 0.00% 0.00% Hex2Cer d18:0/22:2944.7034 0.00% 0.00% Hex2Cer d18:1/22:1; Hex2Cer d18:1/21:2 (1OH)944.7034 19.31% 0.00% Hex2Cer d18:1/22:0; Hex2Cer d18:1/21:1 (1OH)946.7190 19.32% 0.00% Hex2Cer d18:0/22:1; Hex2Cer d18:0/21:2 (1OH)946.7191 19.32% 0.00% Hex2Cer d18:1/21:0 (1OH) 948.6985 19.34% 0.00%Hex2Cer d18:0/22:0; Hex2Cer d18:0/21:1 (1OH) 948.7347 19.34% 0.00%Hex2Cer d18:0/21:0 (1OH) 950.7142 19.35% 0.00% Hex2Cer d18:1/23:2956.7033 0.00% 0.00% Hex2Cer d18:0/23:2 958.7190 0.00% 0.00% Hex2Cerd18:1/23:1; Hex2Cer d18:1/22:2 (1OH) 958.7190 19.95% 0.00% Hex2Cerd18:1/23:0; Hex2Cer d18:1/22:1 (1OH) 960.7346 19.97% 0.00% Hex2Cerd18:0/23:1; Hex2Cer d18:0/22:2 (1OH) 960.7347 19.97% 0.00% Hex2Cerd18:1/22:0 (1OH) 962.7141 19.98% 0.00% Hex2Cer d18:0/23:0; Hex2Cerd18:0/22:1 (1OH) 962.7503 19.98% 0.00% Hex2Cer d18:0/22:0 (1OH) 964.729819.99% 0.00% Hex2Cer d18:1/24:2 970.7190 0.00% 0.00% Hex2Cer d18:1/24:1;Hex2Cer d18:1/23:2 (1OH) 972.7346 20.61% 0.00% Hex2Cer d18:0/24:2972.7347 0.00% 0.00% Hex2Cer d18:0/24:1; Hex2Cer d18:0/23:2 (1OH)974.7503 20.62% 0.00% Hex2Cer d18:1/24:0; Hex2Cer d18:1/23:1 (1OH)974.7503 20.62% 0.00% Hex2Cer d18:1/23:0 (1OH) 976.7298 20.64% 0.00%Hex2Cer d18:0/24:0; Hex2Cer d18:0/23:1 (1OH) 976.7660 20.64% 0.00%Hex2Cer d18:0/23:0 (1OH) 978.7455 20.65% 0.00% Hex2Cer d18:1/25:2984.7346 0.00% 0.00% Hex2Cer d18:0/25:2 986.7503 0.00% 0.00% Hex2Cerd18:1/25:1; Hex2Cer d18:1/24:2 (1OH) 986.7503 21.28% 0.00% Hex2Cerd18:1/25:0; Hex2Cer d18:1/24:1 (1OH) 988.7659 21.29% 0.00% Hex2Cerd18:0/25:1; Hex2Cer d18:0/24:2 (1OH) 988.7660 21.29% 0.00% Hex2Cerd18:1/24:0 (1OH) 990.7454 21.30% 0.00% Hex2Cer d18:0/25:0; Hex2Cerd18:0/24:1 (1OH) 990.7816 21.30% 0.00% Hex2Cer d18:0/24:0 (1OH) 992.761121.32% 0.00% Hex2Cer d18:1/26:2 998.7502 0.00% 0.00% Hex2Cer d18:0/26:21000.7659 0.00% 0.00% Hex2Cer d18:1/26:1; Hex2Cer d18:1/25:2 (1OH)1000.7659 21.96% 0.00% Hex2Cer d18:1/26:0; Hex2Cer d18:1/25:1 (1OH)1002.7815 21.97% 0.00% Hex2Cer d18:0/26:1; Hex2Cer d18:0/25:2 (1OH)1002.7816 21.97% 0.00% Hex2Cer d18:1/25:0 (1OH) 1004.7611 21.98% 0.00%Hex2Cer d18:0/26:0; Hex2Cer d18:0/25:1 (1OH) 1004.7972 21.98% 0.00%Hex2Cer d18:0/25:0 (1OH) 1006.7768 22.00% 0.00% Hex2Cer d18:1/27:21012.7658 0.00% 0.00% Hex2Cer d18:0/27:2 1014.7815 0.00% 0.00% Hex2Cerd18:1/27:1; Hex2Cer d18:1/26:2 (1OH) 1014.7815 22.65% 0.00% Hex2Cerd18:1/27:0; Hex2Cer d18:1/26:1 (1OH) 1016.7971 22.66% 0.00% Hex2Cerd18:0/27:1; Hex2Cer d18:0/26:2 (1OH) 1016.7972 22.66% 0.00% Hex2Cerd18:1/26:0 (1OH) 1018.7767 22.68% 0.00% Hex2Cer d18:0/27:0; Hex2Cerd18:0/26:1 (1OH) 1018.8128 22.68% 0.00% Hex2Cer d18:0/26:0 (1OH)1020.7924 22.69% 0.00% Hex2Cer d18:1/28:2 1026.7815 0.00% 0.00% Hex2Cerd18:1/28:1; Hex2Cer d18:1/27:2 (1OH) 1028.7971 23.35% 0.00% Hex2Cerd18:0/28:2 1028.7972 0.00% 0.00% Hex2Cer d18:0/28:1; Hex2Cer d18:0/27:2(1OH) 1030.8128 23.37% 0.00% Hex2Cer d18:1/28:0; Hex2Cer d18:1/27:1(1OH) 1030.8128 23.37% 0.00% Hex2Cer d18:1/27:0 (1OH) 1032.7924 23.38%0.00% Hex2Cer d18:0/28:0; Hex2Cer d18:0/27:1 (1OH) 1032.8285 23.38%0.00% Hex2Cer d18:0/27:0 (1OH) 1034.8081 23.40% 0.00% Hex2Cerd18:1/29:1; Hex2Cer d18:1/28:2 (1OH) 1042.8128 0.00% 0.00% Hex2Cerd18:1/29:0; Hex2Cer d18:1/28:1 (1OH) 1044.8284 24.08% 0.00% Hex2Cerd18:0/29:1; Hex2Cer d18:0/28:2 (1OH) 1044.8285 0.00% 0.00% Hex2Cerd18:1/28:0 (1OH) 1046.8260 24.10% 0.00% Hex2Cer d18:0/29:0; Hex2Cerd18:0/28:1 (1OH) 1046.8441 24.09% 0.00% Hex2Cer d18:0/28:0 (1OH)1048.8417 24.11% 0.00% Hex2Cer d18:1/30:1; Hex2Cer d18:1/29:2 (1OH)1056.8287 0.00% 0.00% Hex2Cer d18:1/30:0; Hex2Cer d18:1/29:1 (1OH)1058.8440 24.81% 0.00% Hex2Cer d18:0/30:1; Hex2Cer d18:0/29:2 (1OH)1058.8444 0.00% 0.00% Hex2Cer d18:1/29:0 (1OH) 1060.8237 24.83% 0.00%Hex2Cer d18:0/30:0; Hex2Cer d18:0/29:1 (1OH) 1060.8597 24.83% 0.00%Hex2Cer d18:0/29:0 (1OH) 1062.8394 24.84% 0.00% LPE 6:0 314.1363 0.00%0.00% LPE 8:0 342.1676 0.00% 0.00% LPE 10:0 370.1989 0.00% 0.00% LPE12:0 398.2302 0.00% 0.00% LPE 14:0 (IS) 426.2615 0.00% 0.00% LPE P-16:0;LPE O-16:1 438.2979 0.00% 0.00% LPE O-16:0; LPE 15:0 440.2772 3.95%0.00% LPE 16:2 450.2615 0.00% 0.00% LPE 16:1 452.2772 4.15% 0.00% LPE16:0 454.2928 4.16% 0.00% LPE P-18:0; LPE O-18:1; LPE 17:1 466.32980.00% 0.00% LPE O-18:0; LPE 17:0 468.3085 4.49% 0.00% LPE 18:4 474.26150.00% 0.00% LPE 18:3 476.2772 4.68% 0.00% LPE 18:2 478.2928 4.69% 0.00%LPE 18:1 480.3085 4.69% 0.00% LPE 18:0 482.3241 4.70% 0.00% LPE P-20:0;LPE O-20:1 494.3605 0.00% 0.00% LPE O-20:0; LPE 19:0 496.3398 5.08%0.00% LPE 20:5 500.2766 0.00% 0.00% LPE 20:4 502.2923 5.26% 0.00% LPE20:3 504.3079 5.27% 0.00% LPE 20:2 506.3241 5.27% 0.00% LPE 20:1508.3398 5.28% 0.00% LPE 20:0 510.3554 5.29% 0.00% LPE P-22:0; LPEO-22:1 522.3918 0.00% 0.00% LPE O-22:0; LPE 21:0 524.3711 5.71% 0.00%LPE 22:6 526.2928 5.72% 0.00% LPE 22:5 528.3079 5.89% 0.00% LPE 22:4530.3235 5.89% 0.00% LPE 22:1 536.3711 0.00% 0.00% LPE 22:0 538.38675.92% 0.00% LPE P-24:0; LPE O-24:1 550.4231 0.00% 0.00% LPE O-24:0; LPE23:0 552.4024 6.40% 0.00% LPE 24:1 564.4024 0.00% 0.00% LPE 24:0566.4180 6.61% 0.00% LPE 25:0 580.4337 0.00% 0.00% LPE 26:0 594.44930.00% 0.00% LPE 27:0 608.4650 0.00% 0.00% LPE 28:0 622.4806 0.00% 0.00%PE 28:0 636.4599 0.00% 0.00% PE 29:0 650.4755 0.00% 0.00% PE 30:1662.4755 0.00% 0.00% PE 30:0 664.4912 9.17% 0.00% PE P-32:1; PE-3L2674.5119 0.00% 0.00% PE P-32:0; PE-31:1 676.4912 9.83% 0.00% PE O-32:0;PE-31:0 678.5068 9.84% 0.00% PE 32:3; PE P-33:2 686.4755 0.00% 0.00% PE32:2; PE P-33:1 688.4912 10.03% 0.00% PE 32:1; PE P-33:0 690.5068 10.04%0.00% PE 32:0; PE O-33:0 692.5225 10.05% 0.00% PE 32:0 692.5225 10.05%0.00% PE O-34:3/P-34:2 700.5276 0.00% 0.00% PE P-34:1; PE 33:2 702.50680.00% 0.00% PE O-34:2/P-34:1 702.5432 10.75% 0.00% PE P-34:0; PE 33:1704.5225 10.76% 0.00% PE O-34:1/P-34:0 704.5589 10.76% 0.00% PEO-34:0;PE 33:0 706.5381 10.77% 0.00% PE 34:4; PE P-35:3 712.4912 0.00%0.00% PE 34:3; PE P-35:2 714.5068 10.95% 0.00% PE 34:2; PE P-35:1716.5225 10.96% 0.00% PE 34:1; PE P-35:0 718.5381 10.97% 0.00% PE 34:0;PE P-36:6 720.5538 10.98% 0.00% PE P-36:5; PE 35:6 722.4756 11.69% 0.00%PE O-36:6/P-36:5 722.5119 0.00% 0.00% PE P-36:4; PE 35:5 724.4912 11.70%0.00% PE O-36:5/P-36:4 724.5276 11.70% 0.00% PE P-36:3; PE 35:4 726.506811.71% 0.00% PE O-36:4/P-36:3 726.5432 11.71% 0.00% PE P-36:2; PE 35:3728.5225 11.72% 0.00% PE O-36:3/P-36:2 728.5589 11.72% 0.00% PE P-36:1;PE 35:2 730.5381 11.73% 0.00% PE O-36:2/P-36:1 730.5745 11.73% 0.00% PEP-36:0; PE 35:1 732.5538 11.74% 0.00% PE 36:7; PE O-36:0; PE 35:0734.5694 11.75% 0.00% PE 36:6; PE P-37:5 736.4912 11.90% 0.00% PE 36:5;PE P-37:4 738.5068 11.91% 0.00% PE 36:4; PE P-37:3 740.5225 11.92% 0.00%PE 36:3; PE P-37:2 742.5381 11.93% 0.00% PE 36:2; PE P-37:1 744.553811.94% 0.00% PE 36:1; PE P-37:0 746.5694 11.95% 0.00% PE O-38:7/P-38:6748.5276 0.00% 0.00% PE 36:0; PE P-38:6 748.5851 11.96% 0.00% PE P-38:5;PE 37:6 750.5063 12.71% 0.00% PE O-38:6/P-38:5 750.5432 12.71% 0.00% PEP-38:4; PE 37:5 752.5220 14.35% 0.00% PE O-38:5/P-38:4 752.5589 12.72%0.00% PE P-38:3; PE 37:4 754.5376 14.36% 0.00% PE O-38:4/P-38:3 754.574512.73% 0.00% PE P-38:2; PE 37:3 756.5538 14.37% 0.00% PE O-38:3/P-38:2756.5902 12.74% 0.00% PE P-38:1; PE 37:2 758.5694 14.39% 0.00% PEP-38:0; PE 37:1 760.5851 14.40% 0.00% PE 38:7; PE O-38:0; PE 37:0762.6007 14.41% 0.00% PE 38:6; PE P-39:5 764.5225 14.55% 0.00% PE 38:5;PE P-39:4 766.5382 14.56% 0.00% PE 38:4; PE P-40:10; PE P-29:3 768.553814.57% 0.00% PE 38:3; PE P-40:9; PE P-39:2 770.5694 14.586% 0.00% PE38:2; PE P-40:8; PE P-39:1 772.5851 14.60% 0.00% PE 38:1; PE P-40:7; PEP-39:0 774.6007 14.61% 0.00% PE O-40:7/P-40:6 776.5589 0.00% 0.00% PE38:0; PE P-40:6, PE O-39:0 776.6164 14.62% 0.00% PE P-40:5; PE 39:6778.5382 13.78% 0.00% PE O-40:6/P-40:5 778.5745 13.78% 0.00% PE P-40:4;PE 39:5 780.5538 13.79% 0.00% PE O-40:5/P-40:4 780.5902 13.79% 0.00% PEP-40:3; PE 39:4 782.5694 15.51% 0.00% PE 40:10; PE P-40:2; PE 39:3784.4912 15.52% 0.00% PE 40:9; PE P-40:1; PE 39:2 786.5069 15.66% 0.00%PE 40:8; PE P-40:0; PE 39:1 788.5225 15.67% 0.00% PE 40:7; PE O-40:0; PE39:0 790.5381 15.69% 0.00% PE 40:6; PE P-41:5 792.5538 15.70% 0.00% PE40:5; PE P-41:4 794.5695 15.71% 0.00% PE 40:4; PE P-42:10; PE P-41:3796.5851 15.72% 0.00% PE 40:3; PE P-42:9; PE P-41:2 798.6008 15.73%0.00% PE 40:2; PE P-42:8; PE P-41:1 800.6164 15.75% 0.00% PE 40:1; PEP-42:7; PE P-41:0 802.6320 15.76% 0.00% PE 40:0; PE P-42:6, PE O-41:0804.6477 15.77% 0.00% PE P-42:5; PE 41:6 806.5694 14.90% 0.00% PEO-42:6/P-42:5 806.6058 0.00% 0.00% PE P-42:4; PE 41:5 808.5851 16.69%0.00% PE P-42:3; PE 41:4 810.6007 16.71% 0.00% PE 42:10; PE P-42:2; PE41:3 812.5225 16.72% 0.00% PE 42:9; PE P-42:1; PE 41:2 814.5382 16.85%0.00% PE 42:8; PE P-42:0; PE 41:1 816.5538 16.87% 0.00% PE 42:7; PEO-42:0 818.5694 16.88% 0.00% PE 42:6; PE P-43:5 820.5851 16.89% 0.00% PE42:5; PE P-43:4 822.6007 16.91% 0.00% PE 42:4; PE P-43:3 824.6164 16.92%0.00% PE 42:3; PE P-43:2 826.6320 16.93% 0.00% PE 42:2; PE P-43:1828.6477 16.94% 0.00% PE 42:1; PE P-43:0 830.6633 16.96% 0.00% PE 42:0;PE O-43:0 832.6790 16.97% 0.00% PE 44:0 860.7103 0.00% 0.00% LPI 6:0450.1734 0.00% 0.00% LPI 8:0 478.2047 0.00% 0.00% LPI 10:0 506.23600.00% 0.00% LPI 12:0 534.2673 0.00% 0.00% LPI 14:0 562.2986 0.00% 0.00%LPI P-16:0; LPI O-16:1 574.3350 0.00% 0.00% LPI O-16:0; LPI 15:0576.3143 6.16% 0.00% LPI 16:2 586.2986 0.00% 0.00% PI 16:0 587.28270.00% 0.00% LPI 16:1 588.3143 6.36% 0.00% LPI 16:0 590.3299 6.37% 0.00%LPI P-18:0; LPI O-18:1; LPI 17:1 602.3669 0.00% 0.00% PI 16:0; LPIO-18:0; LPI 17:0 (IS) 604.3456 6.80% 0.00% LPI 18:4 610.2986 0.00% 0.00%LPI 18:3 612.3143 6.99% 0.00% LPI 18:2 614.3299 6.99% 0.00% LPI 18:1616.3456 7.00% 0.00% LPI 18:0 618.3612 7.01% 0.00% LPI P-20:0; LPIO-20:1 630.3976 0.00% 0.00% LPI O-20:0; LPI 19:0 632.3769 7.49% 0.00%LPI 20:5 636.3137 0.00% 0.00% LPI 20:4 638.3294 7.67% 0.00% LPI 20:3640.3450 7.68% 0.00% LPI 20:2 642.3612 7.68% 0.00% LPI 20:1 644.37697.69% 0.00% LPI 20:0 646.3925 7.70% 0.00% LPI P-22:0; LPI O-22:1658.4289 0.00% 0.00% LPI O-22:0; LPI 21:0 660.4082 8.23% 0.00% LPI 22:6662.3299 8.23% 0.00% LPI 22:5 664.3450 8.40% 0.00% LPI 22:4 666.36068.41% 0.00% LPI 22:1 672.4082 0.00% 0.00% LPI 22:0 674.4238 8.44% 0.00%LPI P-24:0; LPI O-24:1 686.4602 0.00% 0.00% LPI O-24:0; LPI 23:0688.4395 9.01% 0.00% LPI 24:1 700.4395 0.00% 0.00% LPI 24:0 702.45519.22% 0.00% LPI 25:0 716.4708 0.00% 0.00% LPI 26:0 730.4864 0.00% 0.00%LPI 27:0 744.5021 0.00% 0.00% LPI 28:0 758.5177 0.00% 0.00% PI 28:0772.4970 0.00% 0.00% PI 29:0 786.5126 0.00% 0.00% PI 30:1 798.5126 0.00%0.00% PI 30:0 800.5283 10.04% 0.00% PI P-32:1; PI-31:2 810.5490 0.00%0.00% PI P-32:0; PI-31:1 812.5283 12.85% 0.00% PI O-32:0; PI-31:0814.5440 12.86% 0.00% PI 32:3; PI P-33:2 822.5126 0.00% 0.00% PI 32:2;PI P-33:1 824.5283 13.05% 0.00% PI 32:1; PI P-33:0 826.5440 13.07% 0.00%PI 32:0; PI O-33:0 828.5596 13.08% 0.00% PI P-34:1; PI 33:2 838.54400.00% 0.00% PI P-34:0; PI 33:1 840.5596 13.88% 0.00% PI O-34:0; PI 33:0842.5753 13.90% 0.00% PI 34:4; PI P-35:3 848.5283 0.00% 0.00% PI 34:3;PI P-35:2 850.5440 14.08% 0.00% PI 34:2; PI P-35:1 852.5596 14.09% 0.00%PI 34:1; PI P-35:0 854.5753 14.10% 0.00% PI 34:0; PI P-36:6 856.590914.11% 0.00% PI P-36:5; PI 35:6 858.5127 14.91% 0.00% PI 36:4 859.53310.00% 0.00% PI P-36:4; PI 35:5 860.5283 14.92% 0.00% PI P-36:3; PI 35:4862.5440 14.93% 0.00% PI P-36:2; PI 35:3 864.5596 14.94% 0.00% PIP-36:1; PI 35:2 866.5753 14.95% 0.00% PI P-36:0; PI 35:1 868.5909 14.96%0.00% PI 36:7; PI O-36:0; PI 35:0 870.6066 14.97% 0.00% PI 36:6; PIP-37:5 872.5283 15.12% 0.00% PI 36:5; PI P-37:4 874.5440 15.13% 0.00% PI36:4; PI P-37:3 876.5596 15.14% 0.00% PI 36:3; PI P-37:2 878.5753 15.15%0.00% PI 36:2; PI P-37:1 880.5909 15.16% 0.00% PI 36:1; PI P-37:0882.6066 15.18% 0.00% PI 36:0; PI P-38:6 884.6222 15.19% 0.00% PI 38:5885.5488 0.00% 0.00% PI P-38:5; PI 37:6 886.5434 16.03% 0.00% PI 38:4887.5644 0.00% 0.00% PI P-38:4; PI 37:5 888.5591 16.04% 0.00% PI 38:3889.5801 0.00% 0.00% PI P-38:3; PI 37:4 890.5747 16.06% 0.00% PI P-38:2;PI 37:3 892.5909 16.07% 0.00% PI P-38:1; PI 37:2 894.6066 16.08% 0.00%PI P-38:0; PI 37:1 896.6222 16.09% 0.00% PI 38:7; PI O-38:0; PI 37:0898.6379 16.10% 0.00% PI 38:6; PI P-39:5 900.5596 16.25% 0.00% PI 38:5;PI P-39:4 902.5753 16.26% 0.00% PI 38:4; PI P-40:10; PI P-29:3 904.590916.27% 0.00% PI 38:3; PI P-40:9; PI P-39:2 906.6066 16.28% 0.00% PI38:2; PI P-40:8; PI P-39:1 908.6222 16.29% 0.00% PI 40:7 909.5488 0.00%0.00% PI 38:1; PI P-40:7; PI P-39:0 910.6379 16.31% 0.00% PI 40:6911.5644 0.00% 0.00% PI 38:0; PI P-40:6, PI O-39:0 912.6535 16.32% 0.00%PI P-40:5; PI 39:6 914.5753 17.21% 0.00% PI P-40:4; PI 39:5 916.590917.22% 0.00% PI P-40:3; PI 39:4 918.6066 17.23% 0.00% PI 40:10; PIP-40:2; PI 39:3 920.5283 17.25% 0.00% PI 40:9; PI P-40:1; PI 39:2922.5440 17.38% 0.00% PI 40:8; PIP-40:0; PI 39:1 924.5596 17.40% 0.00%PI 40:7; PI O-40:0; PI 39:0 926.5753 17.41% 0.00% PI 40:6; PI P-41:5928.5909 17.42% 0.00% PI 40:5; PI P-41:4 930.6066 17.43% 0.00% PI 40:4;PI P-42:10; PI P-41:3 932.6222 17.45% 0.00% PI 40:3; PI P-42:9; PIP-41:2 934.6379 17.46% 0.00% PI 40:2; PI P-42:8; PI P-41:1 936.653517.47% 0.00% PI 40:1; PI P-42:7; PI P-41:0 938.6692 17.48% 0.00% PI40:0; PI P-42:6, PI O-41:0 940.6848 17.50% 0.00% PI P-42:5; PI 41:6942.6066 18.43% 0.00% PI P-42:4; PI 41:5 944.6222 18.44% 0.00% PIP-42:3; PI 41:4 946.6379 18.46% 0.00% PI 42:10; PI P-42:2; PI 41:3948.5596 18.47% 0.00% PI 42:9; PIP-42:1; PI 41:2 950.5753 18.61% 0.00%PI 42:8; PI P-42:0; PI 41:1 952.5909 18.62% 0.00% PI 42:7; PI O-42:0954.6066 18.63% 0.00% PI 42:6; PI P-43:5 956.6222 18.65% 0.00% PI 42:5;PI P-43:4 958.6379 18.66% 0.00% PI 42:4; PI P-43:3 960.6535 18.67% 0.00%PI 42:3; PI P-43:2 962.6692 18.68% 0.00% PI 42:2; PI P-43:1 964.684818.70% 0.00% PI 42:1; PI P-43:0 966.7005 18.71% 0.00% PI 42:0; PI O-43:0968.7161 18.72% 0.00% PI 44:0 996.7474 0.00% 0.00% LPC 6:0 356.18330.00% 0.00% LPC 8:0 384.2146 0.00% 0.00% LPC 10:0 412.2459 0.00% 0.00%LPC 12:0 440.2772 0.00% 0.00% LPC 14:0 468.3085 0.00% 0.00% LPC P-16:0;LPC O-16:1 480.3449 0.00% 0.00% LPC O-16:0; LPC 15:0 482.3241 4.78%0.00% LPC 16:2 492.3085 0.00% 0.00% LPC 16:1 494.3241 4.98% 0.00% LPC16:0 496.3398 4.99% 0.00% LPC P-18:0; LPC O-18:1; LPC 17:1 508.37670.00% 0.00% LPC O-18:0; LPC 17:0 (IS) 510.3554 5.39% 0.00% LPC O-18:0510.3918 0.00% 0.00% LPC 18:4 516.3085 0.00% 0.00% LPC 18:3 518.32415.58% 0.00% LPC 18:2 520.3398 5.58% 0.00% LPC 18:1 522.3554 5.59% 0.00%LPC 18:1 522.3554 5.59% 0.00% LPC 18:0 524.3711 5.60% 0.00% LPC P-20:0;LPC O-20:1 536.4075 0.00% 0.00% LPC O-20:0; LPC 19:0 538.3867 6.05%0.00% LPC 20:5 542.3235 0.00% 0.00% LPC 20:4 544.3392 6.23% 0.00% LPC20:3 546.3548 6.24% 0.00% LPC 20:2 548.3711 6.25% 0.00% LPC 20:1550.3867 6.25% 0.00% LPC 20:0 552.4024 6.26% 0.00% LPC P-22:0; LPCO-22:1 564.4388 0.00% 0.00% LPC O-22:0; LPC 21:0 566.4180 6.76% 0.00%LPC 22:6 568.3398 6.77% 0.00% LPC 22:5 570.3548 6.93% 0.00% LPC 22:4572.3704 6.94% 0.00% LPC 22:1 578.4180 0.00% 0.00% LPC 22:0 580.43376.97% 0.00% LPC P-24:0; LPC O-24:1 592.4701 0.00% 0.00% LPC O-24:0; LPC23:0 594.4493 7.52% 0.00% LPC 24:1 606.4493 0.00% 0.00% LPC 24:0608.4650 7.73% 0.00% SM 28:2 617.4653 0.00% 0.00% SM 28:1 619.4809 8.04%0.00% SM 28:0 621.4966 8.05% 0.00% LPC 25:0 622.4806 0.00% 37.46% SM29:2 631.4809 0.00% 0.00% SM 29:1 633.4966 8.45% 0.00% SM 29:0 635.51238.46% 0.00% LPC 26:0 636.4963 0.00% 38.57% SM 30:2 645.4966 0.00% 0.00%SM 30:1 647.5123 8.88% 0.00% SM 30:0 649.5279 8.89% 0.00% LPC 27:0650.5119 0.00% 39.67% SM 31:2 659.5123 0.00% 0.00% SM 31:1 661.52799.32% 0.00% SM 31:0 663.5436 9.33% 0.00% LPC 28:0 664.5276 0.00% 40.78%SM 32:2 673.5279 0.00% 0.00% SM 32:1 675.5436 9.77% 0.00% SM d32:1675.5436 0.00% 0.00% SM 32:0 677.5592 9.78% 0.00% PC 28:0 678.5068 0.00%41.88% SM 33:2 687.5436 0.00% 0.00% SM 33:1 689.5592 10.23% 0.00% PCO-30:1/P-30:0 690.5432 0.00% 0.00% SM 33:0 691.5749 10.24% 0.00% PC 29:0692.5225 0.00% 42.99% PC O-30:0 692.5589 10.30% 0.00% SM 34:2 701.55920.00% 0.00% SM 34:1 703.5749 10.70% 0.00% PC 30:1 704.5225 0.00% 44.07%SM 34:0 705.5905 10.71% 42.64% PC 30:0 706.5381 10.51% 44.09% SM 35:2715.5749 0.00% 0.00% PC P-32:1; PC-31:2 716.5589 0.00% 45.15% PCO-32:2/P-32:1 716.5589 0.00% 0.00% SM 35:1 717.5905 11.19% 44.81% PCP-32:0; PC-31:1 718.5381 11.24% 45.17% PC O-32:1/P-32:0 718.5745 11.24%0.00% SM 35:0 719.6062 11.20% 44.83% PC O-32:0; PC-31:0 720.5538 11.25%45.20% PC O-32:0 720.5902 11.25% 0.00% PC 32:3; PC P-33:2 728.5225 0.00%0.00% SM 36:2 729.5905 0.00% 44.80% PC 32:2; PC P-33:1 730.5381 11.44%46.26% SM 36:1 731.6062 11.69% 44.82% PC 32:1; PC P-33:0 732.5538 11.46%46.28% SM 36:0 733.6218 11.70% 44.85% PC 32:0; PC O-33:0 734.5694 11.47%46.30% PC O-34:3/P-34:2 742.5745 0.00% 0.00% SM 37:2 743.6062 0.00%0.00% PC P-34:1; PC 33:2 744.5538 0.00% 47.36% PC O-34:2/P-34:1 744.590212.23% 0.00% SM 37:1 745.6218 12.20% 47.02% PC P-34:0; PC 33:1 746.569412.25% 47.38% PC O-34:1/P-34:0 746.6058 12.25% 0.00% SM 37:0 747.637512.21% 47.04% PC O-34:0; PC 33:0 748.5851 12.26% 47.41% PC 34:4; PCP-35:3 754.5382 0.00% 0.00% PC 34:3; PC P-35:2 756.5538 12.44% 0.00% SM38:2 757.6218 0.00% 47.01% PC 34:2; PC P-35:1 758.5694 12.45% 48.46% SM38:1 759.6375 12.72% 47.03% PC 34:1; PC P-35:0 760.5851 12.46% 48.49% SM38:0 761.6531 12.74% 47.06% PC 34:0; PC P-36:6 762.6007 12.47% 48.51% PCP-36:5; PC 35:6 764.5226 13.24% 0.00% PC P-36:4; PC 35:5 766.5382 13.25%0.00% PC O-36:5/P-36:4 766.5745 0.00% 0.00% PC P-36:3; PC 35:4 768.553813.26% 0.00% PC O-36:4/P-36:3 768.5902 13.26% 0.00% PC P-36:2; PC 35:3770.5694 13.27% 0.00% PC O-36:3/P-36:2 770.6058 13.27% 0.00% SM 39:2771.6375 0.00% 49.20% PC P-36:1; PC 35:2 772.5851 13.29% 49.57% SM 39:1773.6531 13.26% 49.23% PC P-36:0; PC 35:1 774.6007 13.30% 49.59% SM 39:0775.6688 13.27% 49.25% PC 36:7; PC O-36:0; PC 35:0 776.6164 13.31%49.62% PC 36:6; PC P-37:5 778.5381 13.45% 0.00% PC 36:5; PC P-37:4780.5538 13.46% 0.00% PC 36:4; PC P-37:3 782.5694 13.48% 0.00% SM 40:3783.6375 0.00% 49.20% PC 36:3; PC P-37:2 784.5851 13.49% 50.65% SM 40:2785.6531 13.80% 49.22% PC 36:2; PC P-37:1 786.6007 13.50% 50.67% SM 40:1787.6688 13.81% 49.24% PC 36:1; PC P-37:0 788.6164 13.51% 50.70% SM 40:0789.6844 13.82% 49.26% PC 36:0; PC P-38:6 790.6320 13.52% 50.72% PCP-38:5; PC 37:6 792.5532 14.34% 0.00% PC O-38:6/P-38:5 792.5902 0.00%0.00% PC P-38:4; PC 37:5 794.5689 14.35% 0.00% PC O-38:5/P-38:4 794.605814.35% 0.00% PC P-38:3; PC 37:4 796.5845 14.36% 0.00% PC O-38:4/P-38:3796.6215 14.36% 0.00% PC P-38:2; PC 37:3 798.6007 14.37% 0.00% SM 41:2799.6688 0.00% 51.41% PC P-38:1; PC 37:2 800.6164 14.39% 51.78% SM41:1801.6844 14.37% 51.44% PC P-38:0; PC 37:1 802.6320 14.40% 51.80% SM 41:0803.7001 14.38% 51.46% PC 38:7 804.5538 0.00% 0.00% PC 38:7; PC O-38:0;PC 37:0 804.6477 14.41% 51.82% PC 38:6; PC P-39:5 806.5695 14.55% 0.00%PC 38:5; PC P-39:4 808.5852 14.56% 0.00% PC 38:4; PC P-40:10; PC P-29:3810.6007 14.57% 0.00% SM d42:3 811.6688 0.00% 0.00% PC 38:3; PC P-40:9;PC P-39:2 812.6164 14.59% 0.00% SM 42:2 813.6844 0.00% 51.43% PC 38:2;PC P-40:8; PC P-39:1 814.6320 14.60% 52.88% SM 42:1 815.7001 14.94%51.45% PC 38:1; PC P-40:7; PC P-39:0 816.6477 14.61% 52.91% SM 42:0817.7157 14.95% 51.47% PC O-40:7/P-40:6 818.6058 0.00% 0.00% PC 38:0; PCP-40:6, PC O-39:0 818.6633 14.62% 52.93% PC P-40:5; PC 39:6 820.585215.48% 0.00% PC O-40:6/P-40:5 820.6215 15.48% 0.00% PC P-40:4; PC 39:5822.6008 15.50% 0.00% PC O-40:5/P-40:4 822.6371 15.50% 0.00% PC P-40:3;PC 39:4 824.6164 15.51% 0.00% PC O-40:4/P-40:3 824.6528 15.51% 0.00% PC40:10; PC P-40:2; PC 39:3 826.5382 15.52% 0.00% PC O-40:3/P-40:2826.6684 15.52% 0.00% SM 43:2 827.7001 0.00% 53.48% PC 40:9; PC P-40:1;PC 39:2 828.5539 15.66% 53.99% PC O-40:2/P-40:1 828.6841 15.53% 0.00% SM43:1 829.7157 15.52% 53.50% PC 40:8; PC P-40:0; PC 39:1 830.5695 15.67%54.01% PC O-40:1/P-40:0 830.6997 15.55% 0.00% SM 43:0 831.7314 15.54%53.52% PC 40:7; PC O-40:0; PC 39:0 832.5851 15.69% 54.03% PC 40:6; PCP-41:5 834.6007 15.70% 0.00% PC 40:5; PC P-41:4 836.6165 15.71% 0.00% PC40:4; PC P-42:10; PC P-41:3 838.6321 15.72% 0.00% SM d44:3 839.70010.00% 0.00% PC 40:3; PC P-42:9; PC P-41:2 840.6478 15.73% 0.00% SM 44:2841.7157 0.00% 55.67% PC 40:2; PC P-42:8; PC P-41:1 842.6633 15.75%55.09% SM 44:1 843.7314 16.12% 55.69% PC 40:1; PC P-42:7; PC P-41:0844.6790 15.76% 55.11% SM 44:0 845.7470 16.13% 55.72% PC 40:0; PCP-42:6, PC O-41:0 846.6946 15.77% 55.14% PC P-42:5; PC 41:6 848.616416.68% 0.00% PC O-42:6/P-42:5 848.6528 0.00% 0.00% PC P-42:4; PC 41:5850.6321 16.69% 0.00% PC O-42:5/P-42:4 850.6684 16.69% 0.00% PC P-42:3;PC 41:4 852.6477 16.71% 0.00% PC O-42:4/P-42:3 852.6841 16.71% 0.00% PC42:10; PC P-42:2; PC 41:3 854.5695 16.72% 0.00% PC O-42:3/P-42:2854.6997 16.72% 0.00% PC 42:9; PC P-42:1; PC 41:2 856.5852 16.85% 0.00%PC O-42:2/P-42:1 856.7154 16.73% 0.00% PC 42:8; PC P-42:0; PC 41:1858.6008 16.87% 0.00% PC 42:7; PC O-42:0 860.6164 16.88% 0.00% PC 42:6;PC P-43:5 862.6320 16.89% 0.00% PC 42:5; PC P-43:4 864.6477 16.91% 0.00%PC 42:4; PC P-43:3 866.6633 16.92% 0.00% PC 42:3; PC P-43:2 868.679016.93% 0.00% PC 42:2; PC P-43:1 870.6946 16.94% 0.00% PC 42:1; PC P-43:0872.7103 16.96% 0.00% PC 42:0; PC O-43:0 874.7259 16.97% 0.00% PCO-44:6/P-44:5 876.6841 0.00% 0.00% PC O-44:5/P-44:4 878.6997 17.94%0.00% PC O-44:4/P-44:3 880.7154 17.95% 0.00% PC 44:0 902.7573 0.00%0.00% Sulfatide 30:2 722.4473 0.00% 0.00% Sulfatide 30:1 724.4630 15.10%0.00% Sulfatide 30:0 726.4786 15.11% 0.00% Sulfatide 31:2 736.4630 0.00%0.00% Sulfatide 31:1; Sulfatide 30:2 (1OH) 738.4786 15.55% 0.00%Sulfatide 31:0; Sulfatide 30:1 (1OH) 740.4943 15.56% 0.00% Sulfatide30:0 (1OH) 742.4735 15.57% 0.00% Sulfatide 32:2 750.4786 0.00% 0.00%Sulfatide 32:1; Sulfatide 31:2 (1OH) 752.4946 16.02% 0.00% Sulfatide32:0; Sulfatide 31:1 (1OH) 754.5099 16.03% 0.00% Sulfatide 31:0 (1OH)756.4892 16.04% 0.00% Sulfatide 33:2 764.4943 0.00% 0.00% Sulfatide33:1; Sulfatide 32:2 (1OH) 766.5099 16.50% 0.00% Sulfatide 33:0;Sulfatide 32:1 (1OH) 768.5255 16.51% 0.00% Sulfatide 32:0 (1OH) 770.504816.52% 0.00% Sulfatide 34:2 778.5099 0.00% 0.00% Sulfatide 34:1;Sulfatide 33:2 (1OH) 780.5255 17.00% 0.00% Sulfatide 34:0; Sulfatide33:1 (1OH) 782.5412 17.01% 0.00% Sulfatide 33:0 (1OH) 784.5205 17.02%0.00% Sulfatide 35:2 792.5255 0.00% 0.00% Sulfatide 35:1; Sulfatide 34:2(1OH) 794.5412 17.50% 0.00% Sulfatide 35:0; Sulfatide 34:1 (1OH)796.5569 17.51% 0.00% Sulfatide 34:0 (1OH) 798.5361 17.52% 0.00%Sulfatide 36:2 806.5412 0.00% 0.00% Sulfatide 36:1; Sulfatide 35:2 (1OH)808.5569 18.02% 0.00% Sulfatide 36:0; Sulfatide 35:1 (1OH) 810.572518.03% 0.00% Sulfatide 35:0 (1OH) 812.5518 18.05% 0.00% Sulfatide 37:2820.5569 0.00% 0.00% Sulfatide 37:1; Sulfatide 36:2 (1OH) 822.572518.55% 0.00% Sulfatide 37:0; Sulfatide 36:1 (1OH) 824.5882 18.56% 0.00%Sulfatide 36:0 (1OH) 826.5674 18.57% 0.00% Sulfatide 38:3 832.5569 0.00%0.00% Sulfatide 38:2 834.5725 19.08% 0.00% Sulfatide 38:1; Sulfatide37:2 (1OH) 836.5882 19.09% 0.00% Sulfatide 38:0; Sulfatide 37:1 (1OH)838.6038 19.10% 0.00% Sulfatide 37:0 (1OH) 840.5831 19.11% 0.00%Sulfatide 39:2; Sulfatide 38:3 (1OH) 848.5882 0.00% 0.00% Sulfatide39:1; Sulfatide 38:2 (1OH) 850.6038 19.65% 0.00% Sulfatide 39:0;Sulfatide 38:1 (1OH) 852.6195 19.66% 0.00% Sulfatide 38:0 (1OH) 854.598719.67% 0.00% Sulfatide 40:3 860.5881 0.00% 0.00% Sulfatide 40:2 862.603820.20% 0.00% Sulfatide 40:1; Sulfatide 39:2 (1OH) 864.6195 20.22% 0.00%Sulfatide 40:0; Sulfatide 39:1 (1OH) 866.6351 20.23% 0.00% Sulfatide39:0 (1OH) 868.6144 20.24% 0.00% Sulfatide 41:2; Sulfatide 40:3 (1OH)876.6195 0.00% 0.00% Sulfatide 41:1; Sulfatide 40:2 (1OH) 878.635120.79% 0.00% Sulfatide 41:0; Sulfatide 40:1 (1OH) 880.6508 20.80% 0.00%Sulfatide 40:0 (1OH) 882.6300 20.81% 0.00% Sulfatide 42:3 888.6195 0.00%0.00% Sulfatide 42:2 890.6351 21.37% 0.00% Sulfatide 42:1; Sulfatide41:2 (1OH) 892.6508 21.38% 0.00% Sulfatide 42:0; Sulfatide 41:1 (1OH)894.6664 21.40% 0.00% Sulfatide 41:0 (1OH) 896.6457 21.41% 0.00%Sulfatide 43:2; Sulfatide 42:3 (1OH) 904.6508 0.00% 0.00% Sulfatide43:1; Sulfatide 42:2 (1OH) 906.6664 21.98% 0.00% Sulfatide 43:0;Sulfatide 42:1 (1OH) 908.6821 22.00% 0.00% Sulfatide 42:0 (1OH) 910.661322.01% 0.00% Sulfatide 44:3 916.6508 0.00% 0.00% Sulfatide 44:2 918.666422.59% 0.00% Sulfatide 44:1; Sulfatide 43:2 (1OH) 920.6821 22.60% 0.00%Sulfatide 44:0; Sulfatide 43:1 (1OH) 922.6977 22.62% 0.00% Sulfatide43:0 (1OH) 924.6770 22.63% 0.00% LPA 6:0 288.1206 0.00% 0.00% LPA 8:0316.1519 0.00% 0.00% LPA 10:0 344.1832 0.00% 0.00% LPA 12:0 372.21450.00% 0.00% LPA 14:0 (IS) 400.2458 0.00% 0.00% LPAP-16:0; LPA O-16:1412.2822 0.00% 0.00% LPA O-16:0; LPA 15:0 414.2615 3.46% 0.00% LPA 16:2424.2458 0.00% 0.00% LPA 16:1 426.2615 3.67% 0.00% LPA 16:0 428.27713.67% 0.00% LPA P-18:0; LPA O-18:1; LPA 17:1 440.3140 0.00% 0.00% LPAO-18:0; LPA 17:0 442.2928 3.95% 0.00% LPA 18:4 448.2458 0.00% 0.00% LPA18:3 450.2615 4.15% 0.00% LPA 18:2 452.2771 4.15% 0.00% LPA 18:1454.2928 4.16% 0.00% LPA 18:0 456.3084 4.16% 0.00% LPA P-20:0; LPAO-20:1 468.3448 0.00% 0.00% LPA O-20:0; LPA 19:0 470.3241 4.49% 0.00%LPA 20:5 474.2609 0.00% 0.00% LPA 20:4 476.2766 4.68% 0.00% LPA 20:3478.2922 4.69% 0.00% LPA 20:2 480.3084 4.69% 0.00% LPA 20:1 482.32414.70% 0.00% LPA 20:0 484.3397 4.70% 0.00% LPA P-22:0; LPA O-22:1496.3761 0.00% 0.00% LPA O-22:0; LPA 21:0 498.3554 5.08% 0.00% LPA 22:6500.2771 5.09% 0.00% LPA 22:5 502.2922 5.26% 0.00% LPA 22:4 504.30785.27% 0.00% LPA 22:1 510.3554 0.00% 0.00% LPA 22:0 512.3710 5.29% 0.00%LPA P-24:0; LPA O-24:1 524.4074 0.00% 0.00% LPA O-24:0; LPA 23:0526.3867 5.72% 0.00% LPA 24:1 538.3867 0.00% 0.00% LPA 24:0 540.40235.93% 0.00% LPA 25:0 554.4180 0.00% 0.00% LPA 26:0 568.4336 0.00% 0.00%LPA 27:0 582.4493 0.00% 0.00% LPA 28:0 596.4649 0.00% 0.00% PA 28:0 (IS)610.4442 0.00% 0.00% PA 29:0 624.4598 0.00% 0.00% PA 30:1 636.4598 0.00%0.00% PA 30:0 638.4755 8.35% 0.00% PA P-32:1; PA-31:2 648.4962 0.00%0.00% PA P-32:0; PA-31:1 650.4755 8.96% 0.00% PA O-32:0; PA-31:0652.4911 8.97% 0.00% PA 32:3; PA P-33:2 660.4598 0.00% 0.00% PA 32:2; PAP-33:1 662.4755 9.16% 0.00% PA 32:1; PA P-33:0 664.4911 9.17% 0.00% PA32:0; PA O-33:0 666.5068 9.18% 0.00% PA P-34:1; PA 33:2 676.4911 0.00%0.00% PA P-34:0; PA 33:1 678.5068 9.84% 0.00% PA O-34:0; PA 33:0680.5224 9.85% 0.00% PA 34:4; PA P-35:3 686.4755 0.00% 0.00% PA 34:3; PAP-35:2 688.4911 10.03% 0.00% PA 34:2; PA P-35:1 690.5068 10.04% 0.00% PA34:1; PA P-35:0 692.5224 10.05% 0.00% PA 34:0; PA P-36:6 694.5381 10.06%0.00% PA P-36:5; PA 35:6 696.4599 10.72% 0.00% PA P-36:4; PA 35:5698.4755 10.73% 0.00% PA P-36:3; PA 35:4 700.4911 10.74% 0.00% PAP-36:2; PA 35:3 702.5068 10.75% 0.00% PA P-36:1; PA 35:2 704.5224 10.76%0.00% PA P-36:0; PA 35:1 706.5381 10.77% 0.00% PA 36:7; PA O-36:0; PA35:0 708.5537 10.79% 0.00% PA 36:6; PA P-37:5 710.4755 10.93% 0.00% PA36:5; PA P-37:4 712.4911 10.94% 0.00% PA 36:4; PA P-37:3 714.5068 10.95%0.00% PA 36:3; PA P-37:2 716.5224 10.97% 0.00% PA 36:2; PA P-37:1718.5381 10.98% 0.00% PA 36:1; PA P-37:0 720.5537 10.99% 0.00% PA 36:0;PA P-38:6 722.5694 11.00% 0.00% PA P-38:5; PA 37:6 724.4905 11.70% 0.00%PA P-38:4; PA 37:5 726.5062 11.71% 0.00% PA P-38:3; PA 37:4 728.521811.72% 0.00% PA P-38:2; PA 37:3 730.5381 11.73% 0.00% PA P-38:1; PA 37:2732.5537 11.74% 0.00% PA P-38:0; PA 37:1 734.5694 11.75% 0.00% PA 38:7;PA O-38:0; PA 37:0 736.5850 11.76% 0.00% PA 38:6; PA P-39:5 738.506811.91% 0.00% PA 38:5; PA P-39:4 740.5225 11.92% 0.00% PA 38:4; PAP-40:10; PA P-29:3 742.5381 11.93% 0.00% PA 38:3; PA P-40:9; PA P-39:2744.5537 11.941% 0.00% PA 38:2; PA P-40:8; PA P-39:1 746.5694 11.95%0.00% PA 38:1; PA P-40:7; PA P-39:0 748.5850 11.97% 0.00% PA 38:0; PAP-40:6, PA O-39:0 750.6007 11.98% 0.00% PA P-40:5; PA 39:6 752.522512.72% 0.00% PA P-40:4; PA 39:5 754.5381 12.73% 0.00% PA P-40:3; PA 39:4756.5537 12.74% 0.00% PA 40:10; PA P-40:2; PA 39:3 758.4755 12.76% 0.00%PA 40:9; PA P-40:1; PA 39:2 760.4912 12.90% 0.00% PA 40:8; PA P-40:0; PA39:1 762.5068 12.91% 0.00% PA 40:7; PA O-40:0; PA 39:0 764.5224 12.92%0.00% PA 40:6; PA P-41:5 766.5381 12.93% 0.00% PA 40:5; PA P-41:4768.5538 12.95% 0.00% PA 40:4; PA P-42:10; PA P-41:3 770.5694 12.96%0.00% PA 40:3; PA P-42:9; PA P-41:2 772.5851 12.97% 0.00% PA 40:2; PAP-42:8; PA P-41:1 774.6007 12.98% 0.00% PA 40:1; PA P-42:7; PA P-41:0776.6163 12.99% 0.00% PA 40:0; PA P-42:6, PA O-41:0 778.6320 13.01%0.00% PA P-42:5; PA 41:6 780.5537 13.79% 0.00% PA P-42:4; PA 41:5782.5694 13.81% 0.00% PA P-42:3; PA 41:4 784.5850 13.82% 0.00% PA 42:10;PA P-42:2; PA 41:3 786.5068 13.83% 0.00% PA 42:9; PA P-42:1; PA 41:2788.5225 13.97% 0.00% PA 42:8; PA P-42:0; PA 41:1 790.5381 13.98% 0.00%PA 42:7; PA O-42:0 792.5537 13.99% 0.00% PA 42:6; PA P-43:5 794.569414.01% 0.00% PA 42:5; PA P-43:4 796.5850 14.02% 0.00% PA 42:4; PA P-43:3798.6007 14.03% 0.00% PA 42:3; PA P-43:2 800.6163 14.05% 0.00% PA 42:2;PA P-43:1 802.6320 14.06% 0.00% PA 42:1; PA P-43:0 804.6476 14.07% 0.00%PA 42:0; PA O-43:0 806.6633 14.08% 0.00% PA 44:0 834.6946 0.00% 0.00%S1P d16:1 374.2067 0.00% 0.00% S1P d16:0 376.2223 2.60% 0.00% S1P d17:0386.2067 0.00% 0.00% S1P d17:1 388.2223 2.80% 0.00% S1P d18:2 400.22230.00% 0.00% S1P d18:1 402.2380 3.01% 0.00% S1P d18:0 404.2536 3.02%0.00% S1P d19:1 414.2380 0.00% 0.00% S1P d19:0 416.2531 3.24% 0.00% S1Pd20:1 430.2693 0.00% 0.00% S1P d20:0 432.2849 3.48% 0.00% LPS 6:0358.1261 0.00% 0.00% LPS 8:0 386.1574 0.00% 0.00% LPS 10:0 414.18870.00% 0.00% LPS 12:0 442.2200 0.00% 0.00% LPS 14:0 470.2513 0.00% 0.00%LPS P-16:0; LPS O-16:1 482.2877 0.00% 0.00% LPS O-16:0; LPS 15:0484.2670 4.64% 0.00% LPS 16:2 494.2513 0.00% 0.00% LPS 16:1 496.26704.84% 0.00% LPS 16:0 498.2826 4.85% 0.00% LPS P-18:0; LPS O-18:1; LPS17:1 (IS) 510.3196 0.00% 0.00% LPS O-18:0; LPS 17:0 512.2983 5.20% 0.00%LPS 18:4 518.2513 0.00% 0.00% LPS 18:3 520.2670 5.39% 0.00% LPS 18:2522.2826 5.40% 0.00% LPS 18:1 524.2983 5.40% 0.00% LPS 18:0 526.31395.41% 0.00% LPS P-20:0; LPS O-20:1 538.3503 0.00% 0.00% LPS O-20:0; LPS19:0 540.3296 5.81% 0.00% LPS 20:5 544.2664 0.00% 0.00% LPS 20:4546.2821 6.00% 0.00% LPS 20:3 548.2977 6.00% 0.00% LPS 20:2 550.31396.01% 0.00% LPS 20:1 552.3296 6.02% 0.00% LPS 20:0 554.3452 6.02% 0.00%LPS P-22:0; LPS O-22:1 566.3816 0.00% 0.00% LPS O-22:0; LPS 21:0568.3609 6.48% 0.00% LPS 22:6 570.2826 6.48% 0.00% LPS 22:5 572.29776.65% 0.00% LPS 22:4 574.3133 6.66% 0.00% LPS 22:1 580.3609 0.00% 0.00%LPS 22:0 582.3765 6.69% 0.00% LPS P-24:0; LPS O-24:1 594.4129 0.00%0.00% LPS O-24:0; LPS 23:0 596.3922 7.19% 0.00% LPS 24:1 608.3922 0.00%0.00% LPS 24:0 610.4078 7.40% 0.00% LPS 25:0 624.4235 0.00% 0.00% LPS26:0 638.4391 0.00% 0.00% LPS 27:0 652.4548 0.00% 0.00% LPS 28:0666.4704 0.00% 0.00% PS 28:0 (IS) 680.4497 0.00% 0.00% PS 29:0 694.46530.00% 0.00% PS 30:1 706.4653 0.00% 0.00% PS 30:0 708.4810 10.04% 0.00%PS P-32:1; PS-31:2 718.5017 0.00% 0.00% PS P-32:0; PS-31:1 720.481010.72% 0.00% PS O-32:0; PS-3L0 722.4966 10.73% 0.00% PS 32:3; PS P-33:2730.4653 0.00% 0.00% PS 32:2; PS P-33:1 732.4810 10.92% 0.00% PS 32:1;PS P-33:0 734.4966 10.93% 0.00% PS 32:0; PS O-33:0 736.5123 10.94% 0.00%PS P-34:1; PS 33:2 746.4966 0.00% 0.00% PS P-34:0; PS 33:1 748.512311.68% 0.00% PS O-34:0; PS 33:0 750.5279 11.69% 0.00% PS 34:4; PS P-35:3756.4810 0.00% 0.00% PS 34:3; PS P-35:2 758.4966 11.87% 0.00% PS 34:2;PS P-35:1 760.5123 11.88% 0.00% PS 34:1; PS P-35:0 762.5279 11.89% 0.00%PS 34:0; PS P-36:6 764.5436 11.90% 0.00% PS P-36:5; PS 35:6 766.465412.67% 0.00% PS P-36:4; PS 35:5 768.4810 12.64% 0.00% PS P-36:3; PS 35:4770.4966 12.65% 0.00% PS P-36:2; PS 35:3 772.5123 12.66% 0.00% PSP-36:1; PS 35:2 774.5279 12.67% 0.00% PS P-36:0; PS 35:1 776.5436 12.68%0.00% PS 36:7; PS O-36:0; PS 35:0 778.5593 12.69% 0.00% PS 36:6; PSP-37:5 780.4810 12.84% 0.00% PS 36:5; PS P-37:4 782.4966 12.85% 0.00% PS36:4; PS P-37:3 784.5123 12.86% 0.00% PS 36:3; PS P-37:2 786.5279 12.87%0.00% PS 36:2; PS P-37:1 788.5436 12.88% 0.00% PS 36:1; PS P-37:0790.5593 12.90% 0.00% PS 36:0; PS P-38:6 792.5749 12.91% 0.00% PSP-38:5; PS 37:6 794.4961 13.68% 0.00% PS P-38:4; PS 37:5 796.5118 13.69%0.00% PS P-38:3; PS 37:4 798.5274 13.70% 0.00% PS P-38:2; PS 37:3800.5436 13.71% 0.00% PS P-38:1; PS 37:2 802.5593 13.72% 0.00% PSP-38:0; PS 37:1 804.5749 13.74% 0.00% PS 38:7; PS O-38:0; PS 37:0806.5906 13.75% 0.00% PS 38:6; PS P-39:5 808.5123 13.89% 0.00% PS 38:5;PS P-39:4 810.5280 13.90% 0.00% PS 38:4; PS P-40:10; PS P-29:3 812.543613.91% 0.00% PS 38:3; PS P-40:9; PS P-39:2 814.5593 13.925% 0.00% PS38:2; PS P-40:8; PS P-39:1 816.5749 13.94% 0.00% PS 38:1; PS P-40:7; PSP-39:0 818.5906 13.95% 0.00% PS 38:0; PS P-40:6, PS O-39:0 820.606213.96% 0.00% PS P-40:5; PS 39:6 822.5280 14.78% 0.00% PS P-40:4; PS 39:5824.5436 14.79% 0.00% PS P-40:3; PS 39:4 826.5593 14.80% 0.00% PS 40:10;PS P-40:2; PS 39:3 828.4810 14.81% 0.00% PS 40:9; PS P-40:1; PS 39:2830.4967 14.95% 0.00% PS 40:8; PS P-40:0; PS 39:1 832.5123 14.96% 0.00%PS 40:7; PS O-40:0; PS 39:0 834.5279 14.98% 0.00% PS 40:6; PS P-41:5836.5436 14.99% 0.00% PS 40:5; PS P-41:4 838.5593 15.00% 0.00% PS 40:4;PS P-42:10; PS P-41:3 840.5749 15.01% 0.00% PS 40:3; PS P-42:9; PSP-41:2 842.5906 15.03% 0.00% PS 40:2; PS P-42:8; PS P-41:1 844.606215.04% 0.00% PS 40:1; PS P-42:7; PS P-41:0 846.6219 15.05% 0.00% PS40:0; PS P-42:6, PS O-41:0 848.6375 15.06% 0.00% PS P-42:5; PS 41:6850.5593 15.92% 0.00% PS P-42:4; PS 41:5 852.5749 15.94% 0.00% PSP-42:3; PS 41:4 854.5906 15.95% 0.00% PS 42:10; PS P-42:2; PS 41:3856.5123 15.96% 0.00% PS 42:9; PS P-42:1; PS 41:2 858.5280 16.10% 0.00%PS 42:8; PS P-42:0; PS 41:1 860.5436 16.11% 0.00% PS 42:7; PS O-42:0862.5593 16.12% 0.00% PS 42:6; PS P-43:5 864.5749 16.14% 0.00% PS 42:5;PS P-43:4 866.5906 16.15% 0.00% PS 42:4; PS P-43:3 868.6062 16.16% 0.00%PS 42:3; PS P-43:2 870.6219 16.18% 0.00% PS 42:2; PS P-43:1 872.637516.19% 0.00% PS 42:1; PS P-43:0 874.6532 16.20% 0.00% PS 42:0; PS O-43:0876.6688 16.21% 0.00% PS 44:0 904.7001 0.00% 0.00% LPG 6:0 362.15740.00% 0.00% LPG 8:0 390.1887 0.00% 0.00% LPG 10:0 418.2200 0.00% 0.00%LPG 12:0 446.2513 0.00% 0.00% LPG 14:0 (IS) 474.2826 0.00% 0.00%LPGP-16:0; LPG O-16:1 486.3190 0.00% 0.00% LPG O-16:0; LPG 15:0 488.29834.65% 0.00% LPG 16:2 498.2826 0.00% 0.00% LPG 16:1 500.2983 4.85% 0.00%LPG 16:0 502.3139 4.86% 0.00% LPG P-18:0; LPG O-18:1; LPG 17:1 514.35080.00% 0.00% LPG O-18:0; LPG 17:0 516.3296 5.21% 0.00% LPG 18:4 522.28260.00% 0.00% LPG 18:3 524.2983 5.40% 0.00% LPG 18:2 526.3139 5.41% 0.00%LPG 18:1 528.3296 5.42% 0.00% LPG 18:0 530.3452 5.42% 0.00% LPG P-20:0;LPG O-20:1 542.3816 0.00% 0.00% LPG O-20:0; LPG 19:0 544.3609 5.83%0.00% LPG 20:5 548.2977 0.00% 0.00% LPG 20:4 550.3134 6.01% 0.00% LPG20:3 552.3290 6.02% 0.00% LPG 20:2 554.3452 6.02% 0.00% LPG 20:1556.3609 6.03% 0.00% LPG 20:0 558.3765 6.04% 0.00% LPGP-22:0; LPG O-22:1570.4129 0.00% 0.00% LPG O-22:0; LPG 21:0 572.3922 6.49% 0.00% LPG 22:6574.3139 6.50% 0.00% LPG 22:5 576.3290 6.67% 0.00% LPG 22:4 578.34466.67% 0.00% LPG 22:1 584.3922 0.00% 0.00% LPG 22:0 586.4078 6.70% 0.00%LPG P-24:0; LPG O-24:1 598.4442 0.00% 0.00% LPG O-24:0; LPG 23:0600.4235 7.20% 0.00% LPG 24:1 612.4235 0.00% 0.00% LPG 24:0 614.43917.41% 0.00% LPG 25:0 628.4548 0.00% 0.00% LPG 26:0 642.4704 0.00% 0.00%LPG 27:0 656.4861 0.00% 0.00% LPG 28:0 670.5017 0.00% 0.00% PG 28:0684.4810 0.00% 0.00% PG 29:0 698.4966 0.00% 0.00% PG 30:1 710.4966 0.00%0.00% PG 30:0 712.5123 10.06% 0.00% PG P-32:1; PG-31:2 722.5330 0.00%0.00% PG P-32:0; PG-31:1 724.5123 10.74% 0.00% PG O-32:0; PG-31:0726.5279 10.75% 0.00% PG 32:3; PG P-33:2 734.4966 0.00% 0.00% PG 32:2;PG P-33:1 736.5123 10.94% 0.00% PG 32:1; PG P-33:0 738.5279 10.96% 0.00%PG 32:0; PG O-33:0 740.5436 10.97% 0.00% PG P-34:1; PG 33:2 750.52790.00% 0.00% PG P-34:0; PG 33:1 752.5436 11.70% 0.00% PG O-34:0; PG 33:0754.5592 11.71% 0.00% PG 34:4; PG P-35:3 760.5123 0.00% 0.00% PG 34:3;PG P-35:2 762.5279 11.89% 0.00% PG 34:2; PG P-35:1 764.5436 11.90% 0.00%PG 34:1; PG P-35:0 766.5592 11.91% 0.00% PG 34:0; PG P-36:6 768.574911.92% 0.00% PG P-36:5; PG 35:6 770.4967 12.65% 0.00% PG P-36:4; PG 35:5772.5123 12.66% 0.00% PG P-36:3; PG 35:4 774.5279 12.67% 0.00% PGP-36:2; PG 35:3 776.5436 12.68% 0.00% PG P-36:1; PG 35:2 778.5592 12.69%0.00% PG P-36:0; PG 35:1 780.5749 12.70% 0.00% PG 36:7; PG O-36:0; PG35:0 782.5905 12.72% 0.00% PG 36:6; PG P-37:5 784.5123 12.86% 0.00% PG36:5; PG P-37:4 786.5279 12.87% 0.00% PG 36:4; PG P-37:3 788.5436 12.88%0.00% PG 36:3; PG P-37:2 790.5592 12.90% 0.00% PG 36:2; PG P-37:1792.5749 12.91% 0.00% PG 36:1; PG P-37:0 794.5905 12.92% 0.00% PG 36:0;PG P-38:6 796.6062 12.93% 0.00% PG P-38:5; PG 37:6 798.5273 13.70% 0.00%PG P-38:4; PG 37:5 800.5430 13.71% 0.00% PG P-38:3; PG 37:4 802.558613.72% 0.00% PG P-38:2; PG 37:3 804.5749 13.73% 0.00% PG P-38:1; PG 37:2806.5905 13.75% 0.00% PG P-38:0; PG 37:1 808.6062 13.76% 0.00% PG 38:7;PG O-38:0; PG 37:0 810.6218 13.77% 0.00% PG 38:6; PG P-39:5 812.543613.91% 0.00% PG 38:5; PG P-39:4 814.5593 13.92% 0.00% PG 38:4; PGP-40:10; PG P-29:3 816.5749 13.94% 0.00% PG 38:3; PG P-40:9; PG P-39:2818.5905 13.95% 0.00% PG 38:2; PG P-40:8; PG P-39:1 820.6062 13.96%0.00% PG 38:1; PG P-40:7; PG P-39:0 822.6218 13.97% 0.00% PG 38:0; PGP-40:6, PG O-39:0 824.6375 13.98% 0.00% PG P-40:5; PG 39:6 826.559314.80% 0.00% PG P-40:4; PG 39:5 828.5749 14.81% 0.00% PG P-40:3; PG 39:4830.5905 14.82% 0.00% PG 40:10; PG P-40:2; PG 39:3 832.5123 14.84% 0.00%PG 40:9; PG P-40:1; PG 39:2 834.5280 14.98% 0.00% PG 40:8; PG P-40:0; PG39:1 836.5436 14.99% 0.00% PG 40:7; PG O-40:0; PG 39:0 838.5592 15.00%0.00% PG 40:6; PG P-41:5 840.5749 15.01% 0.00% PG 40:5; PG P-41:4842.5906 15.03% 0.00% PG 40:4; PG P-42:10; PG P-41:3 844.6062 15.04%0.00% PG 40:3; PG P-42:9; PG P-41:2 846.6219 15.05% 0.00% PG 40:2; PGP-42:8; PG P-41:1 848.6375 15.06% 0.00% PG 40:1; PG P-42:7; PG P-41:0850.6531 15.07% 0.00% PG 40:0; PG P-42:6, PG O-41:0 852.6688 15.09%0.00% PG P-42:5; PG 41:6 854.5905 15.96% 0.00% PG P-42:4; PG 41:5856.6062 15.97% 0.00% PG P-42:3; PG 41:4 858.6218 15.99% 0.00% PG 42:10;PG P-42:2; PG 41:3 860.5436 16.00% 0.00% PG 42:9; PG P-42:1; PG 41:2862.5593 16.14% 0.00% PG 42:8; PG P-42:0; PG 41:1 864.5749 16.15% 0.00%PG 42:7; PG O-42:0 866.5905 16.16% 0.00% PG 42:6; PG P-43:5 868.606216.17% 0.00% PG 42:5; PG P-43:4 870.6218 16.19% 0.00% PG 42:4; PG P-43:3872.6375 16.20% 0.00% PG 42:3; PG P-43:2 874.6531 16.21% 0.00% PG 42:2;PG P-43:1 876.6688 16.23% 0.00% PG 42:1; PG P-43:0 878.6844 16.24% 0.00%PG 42:0; PG O-43:0 880.7001 16.25% 0.00% PG 44:0 908.7314 0.00% 0.00% *mass-to-charge (m/z) of [M + H]⁺; [M + Na]⁺; [M + NH₄]⁺; or [M +NH₄—H₂O]⁺ depending on individual lipid (sub)class.

TABLE 20 Database of lipid species monitored during the identificationstep in the negative-ion mode together with the calculation of isotopiccorrection for M + 1 and M + 2 isotopic peaks (so called deisotoping).Isotopic correction Lipid m/z M + 2 M + 1 SulfoHexCer 34:1 778.51450.00% 0.00% SulfoHexCer 34:2 (OH) 792.4937 0.00% 0.00% SulfoHexCer 35:1792.5301 0.00% 0.00% SulfoHexCer 34:1 (OH) 794.5094 17.5% 0.00%SulfoHexCer 35:0 794.5458 17.5% 0.00% SulfoHexCer 34:0 (OH) 796.525017.51%  0.00% SulfoHexCer 36:2 804.5301 0.00% 0.00% SulfoHexCer 36:1806.5458  18% 0.00% SulfoHexCer 36:2 (OH) 820.5250 0.00% 0.00%SulfoHexCer 37:1 820.5614 0.00% 0.00% SulfoHexCer 36:1 (OH) 822.540718.55%  0.00% SulfoHexCer 38:2 832.5614 0.00% 0.00% SulfoHexCer 38:1834.5771 19.01%  0.00% SulfoHexCer 39:1 848.5927 0.00% 0.00% SulfoHexCer38:1 (OH) 850.5720 19.65%  0.00% SulfoHexCer 40:2 860.5927 0.00% 0.00%SulfoHexCer 40:1 862.6084 20.19%  0.00% SulfoHexCer 39:1 (OH) 864.587620.21%  0.00% SulfoHexCer 41:2 874.6084 0.00% 0.00% SulfoHexCer 40:2(OH) 876.5876 20.78%  0.00% SulfoHexCer 41:1 876.6240 20.78%  0.00%SulfoHexCer 40:1 (OH) 878.6033 20.79%  0.00% SulfoHexCer 40:0 (OH)880.6189 20.44%  0.00% SulfoHexCer 42:4 884.5927 0.00% 0.00% SulfoHexCer42:3 886.6084 21.34%  0.00% SulfoHexCer 42:2 888.6240 21.35%  0.00%SulfoHexCer 41:2 (OH) 890.6033 21.36%  0.00% SulfoHexCer 42:1 890.639721.36%  0.00% SulfoHexCer 41:1 (OH) 892.6189 21.37%  0.00% SulfoHexCer41:0 (OH) 894.6346 21.02%  0.00% SulfoHexCer 40:0 (2OH) 896.6138 21.03% 0.00% SulfoHexCer 42:3 (OH) 902.6033 0.00% 0.00% SulfoHexCer 43:2902.6397 0.00% 0.00% SulfoHexCer 42:2 (OH) 904.6189 21.97%  0.00%SulfoHexCer 43:1 904.6553 21.97%  0.00% SulfoHexCer 42:1 (OH) 906.634621.98%  0.00% SulfoHexCer 42:0 (OH) 908.6502 21.61%  0.00% SulfoHexCer41:0 (2OH) 910.6295 21.62%  0.00% SulfoHexCer 44:3 914.6397 0.00% 0.00%SulfoHexCer 44:2 916.6553 22.57%  0.00% SulfoHexCer 43:2 (OH) 918.634622.58%  0.00% SulfoHexCer 44:1 918.6710 22.58%  0.00% SulfoHexCer 43:1(OH) 920.6502 22.59%  0.00% SulfoHexCer 42:1 (2OH) 922.6295 22.21% 0.00% SulfoHexCer 43:0 (OH) 922.6659 22.21%  0.00% SulfoHexCer 42:0(2OH) 924.6451 22.22%  0.00% SulfoHexCer 44:2(OH) 932.6502 0.00% 0.00%SulfoHexCer 44:1(OH) 934.6659 22.81%  0.00% SulfoHexCer 43:1 (2OH)936.6451 22.82%  0.00% SulfoHexCer 44:0 (OH) 936.6815 22.82%  0.00%SulfoHexCer 43:0 (2OH) 938.6608 22.83%  0.00% SulfoHex₂Cer 32:1 912.53600.00% 0.00% SulfoHex₂Cer 33:1 926.5516 0.00% 0.00% SulfoHex₂Cer 34:2938.5516 0.00% 0.00% SulfoHex₂Cer 34:1 940.5673 21.31%  0.00%SulfoHex₂Cer 35:1 954.5829 0.00% 0.00% SulfoHex₂Cer 34:1 (OH) 956.562221.9% 0.00% SulfoHex₂Cer 36:2 966.5829 21.9% 0.00% SulfoHex₂Cer 36:1968.5986  22% 0.00% SulfoHex₂Cer 37:1 982.6142 0.00% 0.00% SulfoHex₂Cer36:1 (OH) 984.5935 23.1% 0.00% SulfoHex₂Cer 38:2 994.6142 0.00% 0.00%SSulfoHex₂Cer 38:1 996.6299 23.71%  0.00% SulfoHex₂Cer 39:1 1010.64550.00% 0.00% SulfoHex₂Cer 38:1 (OH) 1012.6248 24.35%  0.00% SulfoHex₂Cer40:3 1020.6299 0.00% 0.00% SulfoHex₂Cer 40:2 1022.6455 24.96%  0.00%SulfoHex₂Cer 40:1 1024.6612 24.97%  0.00% SulfoHex₂Cer 41:3 1034.645524.97%  0.00% SulfoHex₂Cer 41:2 1036.6612 24.98%  0.00% SulfoHex₂Cer40:2 (OH) 1038.6404 24.99%  0.00% SulfoHex₂Cer 41:1 1038.6768 24.99% 0.00% SulfoHex₂Cer 40:1 (OH) 1040.6561  25% 0.00% SulfoHex₂Cer 40:0 (OH)1042.6717 25.1% 0.00% SulfoHex₂Cer 42:4 1046.6455 0.00% 0.00%SulfoHex₂Cer 42:3 1048.6612 26.27%  0.00% SulfoHex₂Cer 42:2 1050.676826.28%  0.00% SulfoHex₂Cer 42:1 1052.6925 26.28%  0.00% SulfoHex₂Cer42:3 (OH) 1064.6561 0.00% 0.00% SulfoHex₂Cer 43:2 1064.6925 0.00% 0.00%SulfoHex₂Cer 42:2 (OH) 1066.6717 26.98%  0.00% SulfoHex₂Cer 43:11066.7081 26.98%  0.00% SulfoHex₂Cer 42:1 (OH) 1068.6874 26.99%  0.00%SulfoHex₂Cer 42:0 (OH) 1070.7030  27% 0.00% SulfoHex₂Cer 44:4 1074.67700.00% 0.00% SulfoHex₂Cer 44:3 1076.6925 27.63%  0.00% SulfoHex₂Cer 44:21078.7081 27.64%  0.00% SulfoHex₂Cer 43:2 (OH) 1080.6874 27.65%  0.00%SulfoHex₂Cer 44:1 1080.7238 27.65%  0.00% SulfoHex₂Cer 42:1 (2*OH)1084.6823 0.00% 0.00% SulfoHex₂Cer 44:2 (OH) 1094.7030 0.00% 0.00%SulfoHexNAcHex2Cer 34:1 1143.6467 0.00% 0.00% SulfoHexNAcHex2Cer 38:11199.7093 0.00% 0.00% SulfoHexNAcHex2Cer 40:2 1225.7249 0.00% 0.00%SulfoHexNAcHex2Cer 40:1 1227.7406 31.93%  0.00% SulfoHexNAcHex2Cer 41:21239.7406 0.00% 0.00% SulfoHexNAcHex2Cer 41:1 1241.7562 32.69%  0.00%SulfoHexNAcHex2Cer 42:4 1249.7249 0.00% 0.00% SulfoHexNAcHex2Cer 42:31251.7406 33.43%  0.00% SulfoHexNAcHex2Cer 42:2 1253.7562 33.44%  0.00%SulfoHexNAcHex2Cer 42:1 1255.7719 33.45%  0.00% SulfoHexNAcHex2Cer 44:31279.7719 0.00% 0.00% SulfoHexNAcHex2Cer 44:2 1281.7875 0.00% 0.00%SulfoHexNAcHex4Cer 38:1 1523.8149 0.00% 0.00% SulfoHexNAcHex4Cer 40:21549.8306 0.00% 0.00% SulfoHexNAcHex4Cer 40:1 1551.8462 44.15%  0.00%SulfoHexNAcHex4Cer 41:2 1563.8462 0.00% 0.00% SulfoHexNAcHex4Cer 41:11565.8619 45.05%  0.00% SulfoHexNAcHex4Cer 42:3 1575.8462 0.00% 0.00%SulfoHexNAcHex4Cer 42:2 1577.8618 45.95%  0.00% SulfoHexNAcHex4Cer 42:11579.8775 45.97%  0.00% SulfoHexNAcHex4Cer 42:2 (OH) 1593.8568 0.00%0.00% SulfoHexNAcHex4Cer 42:1 (OH) 1595.8724 46.2% 0.00%SulfoHexNAcHex3Cer 40:1 1389.7934 0.00% 0.00% SulfoHexNAcHex3Cer 42:21415.8090 0.00% 0.00% SulfoHexNAcHex3Cer 42:1 1417.8246 39.48%  0.00%SulfoHexNAcHex3Cer 42:2 (OH) 1431.8039 0.00% 0.00% SulfoHexNAcHex3Cer42:1 (OH) 1433.8196 39.72%  0.00% GM3 32:1 1123.6746 0.00% 0.00% GM334:2 1149.6902 0.00% 0.00% GM3 34:1 1151.7059 24.68%  0.00% GM3 34:1(OH) 1167.7008 0.00% 0.00% GM3 36:2 1177.7215 0.00% 0.00% GM3 36:11179.7372 26.1% 0.00% GM3 36:1 (OH) 1195.7321 0.00% 0.00% GM3 38:21205.7528 0.00% 0.00% GM3 38:1 1207.7685 27.6% 0.00% GM3 38:1 (OH)1223.7634 0.00% 0.00% GM3 40:2 1233.7841 0.00% 0.00% GM3 40:1 1235.799829.13%  0.00% GM3 41:2 1247.7998 0.00% 0.00% GM3 41:1 1249.8154 29.91% 0.00% GM3 40:1 (OH) 1251.7947 29.93%  0.00% GM3 40:0 (OH) 1253.810429.38%  0.00% GM3 42:3 1259.7998 0.00% 0.00% GM3 42:2 1261.8154 30.7%0.00% GM3 42:1 1263.8311 30.71%  0.00% GM3 42:3 (OH) 1275.7947 0.00%0.00% GM3 42:2 (OH) 1277.8103 30.9% 0.00% GM3 42:1 (OH) 1279.826030.95%  0.00% PS 34:2 758.4977 0.00% 0.00% PS 34:1 760.5134 11.88% 0.00% PS 36:4 782.4977 0.00% 0.00% PS 36:2 786.5290 0.00% 0.00% PS 36:1788.5447 12.88%  0.00% PS 38:4 810.5290 0.00% 0.00% PS 38:3 812.544713.91%  0.00% LPI 16:0 (IS) 571.2889 0.00% 0.00% LPI 18:1 597.3045 0.00%0.00% LPI 18:0 599.3202 6.87% 0.00% LPI 20:4 619.2889 0.00% 0.00% PI32:1 (IS) 807.5029 0.00% 0.00% PI 32:0 809.5185 12.88%  0.00% PI 34:2833.5185 0.00% 0.00% PI 34:1 835.5342 13.89%  0.00% PI 34:0 837.549813.9% 0.00% PI 35:2 847.5342 0.00% 0.00% PI 35:1 849.5498 14.42%  0.00%PI 36:4 857.5185 0.00% 0.00% PI 36:3 859.5342 14.94%  0.00% PI 36:2861.5498 14.95%  0.00% PI 36:1 863.5655 14.96%  0.00% PI 37:4 871.53420.00% 0.00% PI 37:3 873.5498 15.49%  0.00% PI 38:6 881.5185 0.00% 0.00%PI 38:5 883.5342 16.03%  0.00% PI 38:4 885.5498 16.05%  0.00% PI 38:3887.5655 16.06%  0.00% PI 40:6 909.5498 0.00% 0.00% PI 40:5 911.565517.2% 0.00% PI 40:4 913.5811 17.21%  0.00% PG 32:0 (IS) 721.5025 0.00%0.00% PG 34:4 741.4712 0.00% 0.00% PG 34:3 743.4869 11.7% 0.00% PG 34:2745.5025 11.71%  0.00% PG 34:1 747.5181 11.72%  0.00% PG 35:1 761.53380.00% 0.00% PG 36:4 769.5025 0.00% 0.00% PG 36:3 771.5181 12.69%  0.00%PG 36:2 773.5338 11.7% 0.00% PG 36:1 775.5494 11.71%  0.00% PG 38:5795.5181 0.00% 0.00% PG 38:4 797.5338 13.73%  0.00% *mass-to-charge(m/z) of [M − H]⁻.

Statistical Evaluation

Measured concentrations of individual lipids of all measured subjectsare imported into a statistical software (e.g., SIMCA from Umetrics,Sweden). Proper transformation and scaling are chosen, typicallylogarithmic transformation and Pareto or UV scaling. The scaling andtransformation are based on PCA analysis, where normal distribution ofhealthy and pancreatic cancer patients is desirable. PCA analysis isalso used to find potential outliers, if so, the influence of theoutlier on the model is tested (remove the outlier and check the model)and measurement methods are questioned. If a technical problem in caseof outlier measurement is identified, then this measurement is removedfrom the data set. PCA method is used for finding other influentialfactors, such as gender or age. QC samples should cluster closelytogether in PCA analysis, typically close to the middle of the PCAgraph.

The next step is the use of discrimination analysis (OPLS-DA) for thegroup separation of pancreatic cancer patients and healthy volunteersseparately for males and females. The scaling and transformation isbased on PCA results, but the final model has to be found. Models arefitted on all data together and also on separate strata for influentialcovariates. Different groups of lipids are used to build the model andto explore the influence on the healthy vs. cancer separation. The finalmodel is chosen based on multiple factors as good fit (all known samplesare correctly classified), on the model stability (no too influentialobservations), good prediction ability (by cross-validation performedautomatically and then manually with random groups of observations) andbiological reasoning and resistance to removing unimportant lipids. Thefinal models are used to identify unknown samples, and the sensitivityand specificity are estimated based on these predictions. For thispurpose, the receiver operating characteristic (ROC) curves are plotted,and the area under curve (AUC) is calculated. The model is again testedfor good prediction ability via the final classification. After thislast validation of the model, the most dysregulated lipids areidentified using the S-plot or the loading plot. The limits to identifythe lipid as dysregulated may be, for example, the ones from the S-plotwith p bigger than 0.1 and pcorr bigger than 0.4. For these mostdysregulated lipids, the box-plots comparing the average values in thegroup of pancreatic cancer and healthy volunteers for different strataare used to find exact biological interpretation.

Results of Experiments:

The present invention allows to determine whether the tested patientsuffers from cancer, i.e., to distinguish between a healthy person and aperson suffering from a cancer. Furthermore, the present invention alsoallows to determine the specific type of the cancer (also referred toherein as a localization of the cancer). The determination of thespecific type (or localization) of the cancer is typically done as asecond step, after determination that the tested patient suffers fromcancer.

Typically, the distinction between a healthy person and a personsuffering from a cancer has a high sensitivity and a high specificity(above 80%, or even above 90%). The distinction between specific typesof cancer usually has a lower sensitivity and specificity.

EXAMPLE A: UHPSFC/MS LIPIDOMIC ANALYSIS OF HUMAN PLASMA OF CANCERPATIENTS AND HEALTHY VOLUNTEERS

Body fluid samples of various patients suffering from various types ofcancer (kidney, prostate, and breast) and healthy volunteers wereanalyzed for their lipid profile using UHPSFC/MS. Generally, absoluteand relative concentrations of the lipid species can be used todetermine differences in samples of different health state. In thefollowing, absolute concentrations were used for statistical analysis inorder to visualize differences between sample groups and errors insample preparation or measurements. After centering, transformation andPareto scaling, PCA of all samples were performed in order to identifyoutliers and measurement errors.

FIG. 1 shows the results of the PCA analysis of 170 samples of healthyvolunteers, 282 samples of patients suffering from cancer and 38 samplesof unknown classification.

If unsupervised PCA does not show any significant clustering accordingto undesirable parameters (e.g., day of analysis) and unexplainedoutliers, then supervised OPLS-DA analysis is performed to improve thestatistical model applicable also for predictions. If unwantedclustering or outliers are identified, then further investigations arenecessary to identify the reason of those.

For OPLS-DA analysis, samples have to be defined according to theirclassification, i.e., groups of non-cancerous and cancerous samples.OPLS-DA analysis allows the visualization of differences between samplegroups. It is preferred to have a clear differentiation, which means agap between both groups. However, it may happen that there is a smalloverlay of both groups, as differences in the lipid pattern inbiological fluids are not large enough due to the biological variabilityof samples or the investigated cancer type does not show a sufficientlylarge difference in the lipid profile measured in the biological fluid.Generally, it is expected that depending on the cancer type as well ascancer stage, the extent of differences in the lipid profile is altered.

FIG. 2 shows the OPLS-DA analysis of healthy (N, blue) and cancersamples (T) for both genders.

FIG. 2 shows that non-cancerous and cancerous samples can bedifferentiated. However, there is a certain region of overlap of bothsample types with false positives and false negatives, which must bealways expected in the analysis of real-life biological samples. Inorder to see the lipid species responsible for differentiation, anS-plot is prepared (FIG. 3). The S-plot allows the visualization of themost up-(upper right corner of the graph) and down- (bottom left corner)regulated lipid species for all investigated samples.

The prediction of the model encounts all components building up themodel not only the most abundant once shown in the OPLS-DA plot.

The sensitivity describes the prediction power of the model to correctlypredict samples as cancerous samples, whereby the specificity describesthe correctly healthy predicted samples. For instance for the OPLS-DAmodel including healthy and cancer samples for all genders: in total 282cancer samples are included and from those 252 samples were predicted ascancer samples whereas 30 samples were predicted as healthy samples,which leads to a sensitivity of 89.4%. 170 healthy samples are includedin the model, and 137 samples were predicted as healthy, whereby 33samples as cancer samples, which corresponds to a specificity of 80.6%.The sensitivity as well as the specificity may improve, when thestatistical analysis is performed for both genders separately and foreach cancer type separately. FIG. 4 shows the OPLS-DA plot fornon-cancerous and cancerous samples for males and various cancer typesThe statistical analysis of male samples for various cancer types givesthe sensitivity of 93.0% and the specificity of 81.3%.

Compared to results for both genders, the sensitivity increases from89.4 to 93.0% and specificity increases from 80.6 to 81.3%.

The statistical analysis of each cancer type separately in comparison tohealthy samples may improve the sensitivity and specificity. Forexample, FIG. 5 shows the OPLS-DA plot for the analysis of samples ofnon-cancerous and cancerous samples suffering from breast cancer formales.

The differentiation of non-cancerous and cancerous subjects sufferingfrom breast cancer for males is pronounced. However, one has to keep inmind that just a very small amount of breast cancer samples for malesare included in the model, as we did not get more samples provided fromthe hospital. The sensitivity and specificity is 100%.

FIG. 6 shows the OPLS-DA plot for the analysis of non-cancerous andcancerous samples suffering from prostate cancer for males. Thesensitivity for the analysis of prostate cancer is 86.6% and thespecificity is 94.7%.

FIG. 7 shows the OPLS-DA plot for the analysis of non-cancerous andcancerous samples suffering from kidney cancer for males. Thesensitivity for the analysis of kidney cancer samples from males is90.1% and the specificity is 90.7%.

FIG. 8 shows the OPLS-DA plot for the analysis of non-cancerous andcancerous samples for females. The sensitivity for the analysis ofhealthy and cancer samples from females is 88.0% and the specificity is87.4%.

FIG. 9 represents the OPLS-DA plot for the analysis of non-cancerous andcancerous samples suffering from breast cancer samples for females. Thesensitivity is 87.2% and the specificity is 93.7% for the analysis ofbreast cancer samples from females.

FIG. 10 represents the OPLS-DA plot for the analysis of non-cancerousand cancerous samples suffering from kidney cancer for females. Thesensitivity is 74.2% and the specificity is 100% for the analysis ofkidney cancer samples from females.

EXAMPLE B: MALDI-MS LIPIDOMIC ANALYSIS OF BODY FLUIDS (PLASMA, URINE) OFKIDNEY CANCER PATIENTS AND HEALTHY VOLUNTEERS

In this example, body fluid samples (plasma, urine) of various patientssuffering from kidney cancer and healthy volunteers were analyzed fortheir lipid profile using MALDI-MS. In case of plasma samples, absoluteconcentrations (normalization to internal standard) were used for thestatistical analysis in order to visualize differences between samplegroups and errors in sample preparation or measurements. On thecontrary, relative concentrations were used for this purpose in case ofurine samples. Illustrated statistical models for plasma samples includefollowing 74 variables: SM 32:1, SM 33:1, SM 34:2, SM 34:1, SM 34:0, SM35:1, SM 36:2, SM 36:1, SM 36:0, SM 37:1, SM 38:2, SM 38:1, SM 39:2, SM39:1, SM 40:3, SM 40:2, SM 40:1, SM 41:3, SM 41:2, SM 41:1, SM 42:3, SM42:2, SM 42:1, SM 43:3, SM 43:2, SM 43:1, Sul 32:1 (OH), Sul 34:2, Sul34:1, Sul 34:2 (OH), Sul 34:1 (OH), Sul 34:0 (OH), Sul 36:1, Sul 36:1(OH), Sul 38:2, Sul 38:1, Sul 38:2 (OH), Sul 38:1 (OH), Sul 40:2, Sul40:1, Sul 41:2, Sul 40:2 (OH), Sul 41:1, Su 140:1 (OH), Sul 40:0 (OH),Sul 42:3, Sul 42:2, Sul 41:2 (OH), Sul 42:1, Sul 41:1 (OH), Sul 40:0(20H), Sul 42:3 (OH), Sul 42:2 (OH), Sul 42:1 (OH), Sul 42:2 (20H), Sul42:1 (20H), Sul 42:0 (20H), SulfoHex2Cer 42:2, PI 32:1, PI 32:0, PI34:2, PI 34:1, PI 36:4, PI 36:3, PI 36:2, PI 36:1, PI 38:6, PI 38:5, PI38:4, PI 38:3, PI 38:2, PI 40:6, PI 40:5, PI 40:4). Illustratedstatistical models for urine samples include following 46 variables:hydroxypregnenolone sulfate, C21H34O7S sulfate, C21H34O8S sulfate,cortisol sulfate, lithocholic acid sulfate, cholesterol sulfate,glycochenodeoxycholic acid sulfate, taurolithocholic acid,taurodeoxycholic acid, sulfoglycolithocholic acid, taurocholic acid,glycochenodeoxycholic acid sulfate, Sul 34:1, Sul 34:1 (OH), Sul 36:1(OH), Sul 38:1, Sul 38:1 (OH), Sul 40:2, Sul 40:1, Sul 40:2 (OH), Sul41:1, Sul 40:1 (OH), Sul 40:0 (OH), Sul 42:2, Sul 41:2 (OH), Sul 42:1,Sul 41:1 (OH), Sul 41:0 (OH), Sul 40:0 (20H), Sul 42:2 (OH), Sul 42:1(OH), Sul 42:0 (OH), Sul 41:0 (20H), Sul 43:1 (OH), Sul 42:1 (20H), Sul42:0 (20H), Sul 43:0 (20H), SulfoHex2Cer 34:1, SulfoHex2Cer 38:1,SulfoHex2Cer 40:1, SulfoHex2Cer 40:1 (OH), SulfoHex2Cer 42:2,SulfoHex2Cer 42:1, SulfoHex2Cer 40:0 (20H), SulfoHex2Cer 42:1 (OH),SulfoHex2Cer 42:0 (20H).

FIG. 11 shows the results of the PCA of 170 plasma samples of healthyvolunteers (N), 111 plasma samples of patients suffering from kidneycancer (T) and 24 plasma samples of quality control pooled samples (Q)for both genders. Group separation between cancerous and healthy samplesis evident even from unsupervised PCA model, but much better groupseparation is achieved using supervised OPLS-DA model illustrated inFIG. 12, which provides sensitivity 76.6%, specificity 95.3% andaccuracy 87.9%. The sensitivity as well as the specificity is furtherimproved, when the statistical analysis is performed for particulargenders separately as illustrated in FIGS. 13 and 14. The statisticalanalysis of plasma samples of 80 patients suffering from kidney cancerand 75 healthy volunteers (control) gives the sensitivity 90%,specificity 93.3% (accuracy 91.6%) in case of males. The statisticalanalysis of plasma samples of 32 patients suffering from kidney cancerand 95 healthy volunteers gives the sensitivity 83.9%, specificity 98.9%(accuracy 95.2%) for females. Improvement of cancer prediction abilityafter gender separation is also observed for urine samples. FIG. 15shows the results of the PCA analysis of 70 urine samples of healthyvolunteers (34 of males and 36 females), 101 urine samples of patientssuffering from kidney cancer (72 of males and 29 of females) and 16urine samples of quality control pooled samples (Q) for both genders.Corresponding OPLS-DA model for both genders is illustrated in FIG. 16and this model provides sensitivity 87.1%, specificity 82.9% (accuracy85.4%). Subsequent separation to models for individual genders resultsin sensitivity 93.1%, specificity 91.2% (accuracy 92.5%) for males (FIG.17) and sensitivity 86.2%, specificity 88.9% (accuracy 87.7%) forfemales (FIG. 18).

TABLE 21 Statistical parameters for UHPSFC/MS analysis of non- cancerousplasma samples and cancerous plasma samples of males (M) and females (F)for breast cancer. Fold change P-value T-value Species F M F M F M TG48:2 0.9 0.5 2.4E−01 3.2E−02 0.7 2.1 TG 50:3 0.9 0.7 2.0E−01 2.6E−02 0.82.2 TG 50:2 0.9 0.8 2.4E−01 9.1E−02 0.7 1.5 TG 51:4 0.8 0.7 6.5E−021.3E−01 1.5 1.3 TG 51:3 0.7 0.8 1.1E−03 1.1E−01 3.1 1.4 TG 51:2 0.7 0.84.6E−04 1.7E−01 3.4 1.0 TG 52:3 0.9 0.9 2.1E−01 1.7E−01 0.8 1.0 TG 53:40.8 0.8 1.5E−02 1.6E−01 2.2 1.1 TG 53:3 0.7 0.9 2.6E−04 3.8E−01 3.5 0.3Cer40:1 0.5 0.5 1.0E−08 2.9E−04 5.9 4.3 Cer42:1 0.5 0.4 1.4E−10 2.5E−086.7 7.0 PC 32:2 0.5 0.3 6.3E−06 4.5E−08 4.5 6.8 PC 34:2 0.6 0.6 4.2E−101.5E−06 6.5 6.7 PC 34:1 0.6 0.7 9.8E−10 4.6E−03 6.3 3.2 PC O-36:4 0.50.3 7.6E−11 3.5E−09 6.8 8.2 PC O-36:3 0.5 0.4 1.6E−09 1.2E−11 6.3 8.6 PC36:4 0.6 0.5 1.8E−10 1.3E−06 6.7 6.6 PC 36:3 0.6 0.5 1.6E−11 3.9E−11 7.19.4 PC 36:2 0.6 0.5 1.5E−09 4.2E−06 6.3 6.3 PC 38:6 0.6 0.5 3.0E−061.1E−04 4.7 4.5 PC 38:5 0.6 0.4 1.4E−07 6.6E−08 5.4 7.0 PC 38:4 0.7 0.61.6E−07 1.2E−03 5.3 4.0 PC 38:3 0.6 0.5 3.9E−05 1.2E−03 4.1 3.9 SM 34:20.6 0.5 1.6E−10 5.9E−05 6.7 5.4 SM 34:1 0.6 0.5 1.2E−09 3.0E−04 6.3 4.6SM 36:2 0.6 0.6 5.0E−09 2.3E−03 6.0 3.6 SM 36:1 0.6 0.6 2.9E−08 6.4E−035.7 3.1 SM 38:2 0.6 0.6 2.1E−09 4.6E−04 6.2 4.1 SM 38:1 0.5 0.4 8.0E−139.9E−10 7.6 8.8 SM 40:2 0.5 0.4 1.1E−11 1.0E−07 7.2 7.6 SM 40:1 0.5 0.43.2E−12 8.8E−08 7.4 7.9 SM 41:2 0.5 0.4 2.6E−13 8.1E−06 7.8 6.0 SM 41:10.5 0.3 5.2E−14 3.6E−08 8.1 7.8 SM 42:3 0.6 0.6 2.1E−08 1.2E−03 5.8 3.9SM 42:2 0.6 0.5 9.6E−08 3.2E−04 5.5 4.6 SM 42:1 0.3 0.1 1.2E−09 7.3E−156.4 9.5 LPC 16:0 0.6 0.3 1.3E−09 6.3E−12 6.3 12.1 LPC 18:2 0.5 0.23.9E−08 8.8E−18 5.6 12.2 LPC 18:1 0.5 0.2 3.0E−10 1.2E−18 6.6 12.3 LPC18:0 0.5 0.2 3.1E−11 8.6E−16 7.0 14.1

TABLE 22 Statistical parameters for UHPSFC/MS analysis of non- cancerousplasma samples and cancerous plasma samples of males (M) and females (F)for kidney cancer. Fold change P-value T-value Species F M F M F M TG48:2 0.4 0.5 1.0E−07 7.7E−04 5.5 3.3 TG 50:3 0.6 0.6 2.2E−07 4.9E−06 5.54.6 TG 50:2 0.6 0.6 1.0E−07 1.2E−04 5.6 3.8 TG 51:4 0.5 0.6 1.3E−051.6E−05 4.5 4.3 TG 51:3 0.6 0.6 9.4E−08 8.6E−07 5.8 5.1 TG 51:2 0.5 0.51.2E−08 1.0E−05 6.1 4.5 TG 52:3 0.7 0.7 1.3E−04 2.4E−06 3.9 4.8 TG 53:40.6 0.6 4.5E−05 2.3E−05 4.2 4.3 TG 53:3 0.6 0.6 9.8E−06 2.9E−05 4.6 4.2Cer40:1 0.6 0.7 2.8E−04 3.4E−03 3.7 2.8 Cer42:1 0.5 0.6 1.0E−06 1.8E−045.3 3.7 PC 32:2 0.4 0.7 8.3E−07 1.1E−02 5.1 2.3 PC 34:2 0.6 0.7 7.6E−096.6E−07 6.7 5.1 PC 34:1 0.6 0.7 1.9E−06 4.0E−03 5.2 2.7 PC O-36:4 0.70.8 2.4E−03 2.0E−02 3.0 2.1 PC O-36:3 0.6 0.8 8.1E−06 6.4E−02 4.6 1.5 PC36:4 0.7 0.7 1.6E−04 5.9E−04 3.9 3.3 PC 36:3 0.6 0.7 8.9E−08 3.3E−06 6.04.7 PC 36:2 0.6 0.6 6.7E−08 1.6E−07 6.1 5.4 PC 38:6 0.7 0.8 2.3E−027.4E−02 2.1 1.5 PC 38:5 0.7 0.7 1.2E−02 8.1E−03 2.4 2.4 PC 38:4 0.8 0.71.1E−02 3.0E−05 2.4 4.2 PC 38:3 0.7 0.7 4.7E−03 9.2E−05 2.7 3.9 SM 34:20.7 0.7 3.3E−03 1.3E−04 2.9 3.8 SM 34:1 0.6 0.6 2.6E−06 4.8E−09 5.1 6.1SM 36:2 0.8 0.7 3.0E−02 1.9E−03 2.0 3.0 SM 36:1 0.8 0.7 6.9E−02 1.9E−031.5 3.0 SM 38:2 0.7 0.8 1.1E−02 1.8E−02 2.4 2.1 SM 38:1 0.6 0.6 4.7E−057.2E−06 4.3 4.5 SM 40:2 0.6 0.6 2.3E−04 1.2E−05 3.8 4.4 SM 40:1 0.6 0.61.0E−04 4.7E−07 4.1 5.2 SM 41:2 0.5 0.6 4.9E−06 4.6E−06 5.0 4.6 SM 41:10.5 0.5 3.5E−07 3.0E−08 5.6 5.7 SM 42:3 0.8 0.7 4.2E−02 4.3E−03 1.8 2.7SM 42:2 0.7 0.6 3.7E−03 1.7E−05 2.9 4.3 SM 42:1 0.4 0.5 4.7E−05 2.3E−034.2 2.9 LPC 16:0 0.7 0.7 8.9E−03 4.9E−04 2.5 3.4 LPC 18:2 0.4 0.51.2E−08 8.7E−07 6.1 5.0 LPC 18:1 0.6 0.8 7.1E−04 2.3E−02 3.4 2.0 LPC18:0 0.7 0.7 2.5E−02 3.4E−04 2.0 3.5

TABLE 23 Statistical parameters for UHPSFC/MS analysis of non- cancerousplasma samples and cancerous plasma samples of males (M) and females (F)for prostate cancer. Fold change P-value T-value Species M M M TG 48:20.6 7.6E−03 2.5 TG 50:3 0.7 3.0E−04 3.5 TG 50:2 0.7 1.2E−03 3.1 TG 51:40.7 3.3E−03 2.8 TG 51:3 0.7 1.4E−03 3.1 TG 51:2 0.7 3.6E−03 2.7 TG 52:30.7 4.5E−05 4.1 TG 53:4 0.7 4.3E−03 2.7 TG 53:3 0.7 3.4E−03 2.8 Cer40:10.5 3.6E−07 5.2 Cer42:1 0.5 2.0E−08 5.9 PC 32:2 0.6 1.2E−05 4.4 PC 34:20.6 1.3E−13 8.2 PC 34:1 0.6 1.1E−06 5.0 PC 0-36:4 0.5 1.7E−08 5.9 PC0-36:3 0.6 6.2E−06 4.6 PC 36:4 0.6 8.5E−09 6.0 PC 36:3 0.6 7.3E−12 7.4PC 36:2 0.6 1.1E−11 7.3 PC 38:6 0.7 1.5E−04 3.7 PC 38:5 0.7 1.3E−05 4.4PC 38:4 0.6 1.9E−08 5.9 PC 38:3 0.6 8.7E−06 4.5 SM 34:2 0.5 4.5E−10 6.6SM 34:1 0.5 1.3E−13 8.2 SM 36:2 0.6 1.5E−06 4.9 SM 36:1 0.6 2.4E−07 5.3SM 38:2 0.6 2.5E−04 3.6 SM 38:1 0.5 5.2E−11 7.0 SM 40:2 0.5 1.4E−11 7.3SM 40:1 0.4 5.4E−14 8.3 SM 41:2 0.4 6.7E−12 7.4 SM 41:1 0.4 1.0E−13 8.2SM 42:3 0.5 6.4E−10 6.6 SM 42:2 0.5 2.1E−12 7.7 SM 42:1 0.3 1.2E−07 5.5LPC 16:0 0.5 1.0E−15 9.0 LPC 18:2 0.4 1.6E−13 8.2 LPC 18:1 0.5 5.7E−117.0 LPC 18:0 0.5 3.2E−15 8.8

TABLE 24 Statistical parameters for MALDI-MS analysis of non- cancerousplasma samples and cancerous plasma samples of males (M) and females (F)for breast cancer. Fold change P-value T-value Species F M F M F Mcholesterol 0.62 0.73 2.1E−09 6.8E−03 6.2 2.9 sulfate SM 32:1 0.57 0.501.5E−16 1.9E−06 9.0 7.5 SM 33:1 0.54 0.60 9.2E−18 1.3E−03 9.5 4.1 SM34:2 0.66 0.71 1.3E−10 2.4E−03 6.7 3.5 SM 34:1 0.63 0.69 9.8E−15 6.9E−038.4 3.1 SM 34:0 0.60 0.55 3.6E−15 4.5E−04 8.6 4.8 SM 35:1 0.58 0.652.4E−14 3.0E−03 8.2 3.6 SM 36:1 0.67 0.70 2.7E−11 1.7E−02 7.0 2.6 SM37:1 0.57 0.60 5.4E−15 2.9E−03 8.4 3.6 SM 38:2 0.65 0.64 1.6E−10 2.3E−046.7 4.6 SM 38:1 0.61 0.54 2.3E−16 4.4E−05 9.0 6.1 SM 39:2 0.54 0.585.8E−08 4.7E−04 5.6 4.3 SM 39:1 0.52 0.47 7.2E−18 3.0E−05 9.5 6.3 SM40:3 0.52 0.67 4.2E−10 1.6E−02 6.5 2.5 SM 40:2 0.60 0.59 4.3E−15 4.1E−058.5 5.6 SM 40:1 0.60 0.54 5.4E−16 5.2E−05 8.9 6.0 SM 41:2 0.58 0.632.2E−15 1.1E−03 8.6 4.1 SM 41:1 0.58 0.53 4.4E−16 1.4E−04 8.9 5.5 SM42:1 0.61 0.50 2.0E−14 7.5E−06 8.3 7.0 SM 43:1 0.41 0.32 5.4E−12 2.0E−077.5 6.7 Sul 34:2 (OH) 0.57 0.36 9.7E−09 1.0E−08 5.9 7.4 Sul 34:1 (OH)0.63 0.54 8.8E−11 1.4E−05 6.8 5.8 Sul 34:0 (OH) 0.44 0.26 2.7E−135.6E−10 7.8 8.5 Sul 40:2 0.53 0.37 3.8E−08 4.2E−07 5.6 5.6 Sul 40:1 0.540.25 4.4E−11 2.3E−09 6.9 9.3 Sul 40:2 (OH) 0.58 0.34 1.5E−06 1.2E−07 4.86.4 Sul 41:1 0.48 0.34 3.3E−12 5.3E−13 7.4 8.6 Sul 40:1 (OH) 0.59 0.396.5E−11 3.8E−06 6.8 7.0 Sul 42:3 0.58 0.43 2.9E−07 1.5E−05 5.2 4.8 Sul42:2 0.62 0.41 2.3E−10 1.1E−06 6.6 6.7 Sul 42:1 0.52 0.25 3.1E−126.3E−13 7.4 10.1 Sul 41:1 (OH) 0.54 0.34 4.2E−12 3.1E−06 7.3 7.0 Sul42:1 (OH) 0.63 0.39 7.9E−11 2.6E−07 6.8 8.3 Sul 42:1 (2OH) 0.54 0.442.9E−07 1.2E−03 5.2 3.9 PI 34:1 0.61 0.62 1.9E−06 5.0E−03 4.8 3.2 PI36:4 0.63 0.54 3.4E−06 1.6E−03 4.7 3.7 PI 36:3 0.64 0.51 2.0E−05 3.0E−054.2 5.0 PI 36:1 0.58 0.59 9.4E−09 6.9E−03 5.9 3.1 PI 38:5 0.58 0.519.1E−08 1.2E−03 5.5 3.9 PI 38:3 0.65 0.40 7.0E−07 9.2E−09 5.0 8.8

TABLE 25 Statistical parameters for MALDI-MS analysis of non- cancerousplasma samples and cancerous plasma samples of males (M) and females (F)for kidney cancer. Fold change P-value T-value Species F M F M F Mcholesterol 0.57 0.80 5.1E−09 1.6E−02 6.3 2.2 sulfate SM 32:1 0.54 0.604.3E−10 1.4E−11 7.4 7.2 SM 33:1 0.57 0.63 1.4E−08 4.4E−10 6.6 6.6 SM34:2 0.77 0.71 3.7E−03 6.7E−06 2.9 4.5 SM 34:1 0.72 0.75 3.4E−04 3.0E−053.7 4.2 SM 34:0 0.68 0.70 7.8E−05 1.2E−06 4.2 5.0 SM 35:1 0.64 0.661.1E−05 4.1E−08 4.8 5.7 SM 36:2 0.86 0.74 7.2E−02 1.8E−04 1.5 3.7 SM36:1 0.81 0.74 1.5E−02 2.5E−05 2.3 4.2 SM 37:1 0.66 0.65 7.0E−05 1.1E−074.2 5.5 SM 38:2 0.76 0.69 2.7E−03 4.0E−06 3.0 4.7 SM 38:1 0.65 0.641.8E−05 9.9E−10 4.7 6.5 SM 39:1 0.52 0.53 3.4E−08 2.1E−14 6.4 8.4 SM40:2 0.65 0.64 1.0E−05 2.2E−08 4.8 5.8 SM 40:1 0.65 0.63 2.5E−05 6.1E−104.6 6.5 SM 41:2 0.60 0.64 5.2E−07 1.3E−08 5.6 5.9 SM 41:1 0.58 0.591.3E−06 2.8E−12 5.5 7.5 SM 42:1 0.61 0.62 4.6E−06 9.0E−10 5.1 6.5 SM43:3 0.65 0.75 2.1E−06 3.4E−05 5.0 4.1 SM 43:2 0.62 0.64 3.1E−05 2.9E−064.4 4.7 SM 43:1 0.39 0.39 8.2E−10 1.8E−11 6.6 7.4 Sul 32:1 (OH) 0.630.81 6.0E−07 6.9E−04 5.2 3.3 Sul 34:1 0.62 0.68 5.6E−05 2.0E−05 4.3 4.3Sul 34:1 (OH) 0.61 0.68 1.6E−05 2.9E−05 4.7 4.2 Sul 34:0 (OH) 0.43 0.584.9E−08 3.5E−05 6.1 4.1 Sul 40:1 0.62 0.60 4.6E−04 1.1E−05 3.6 4.4 Sul41:1 0.49 0.60 4.2E−07 2.7E−05 5.5 4.2 Sul 40:1 (OH) 0.57 0.58 9.1E−065.1E−08 4.8 5.6 Sul 41:1 (OH) 0.50 0.52 3.6E−07 6.4E−09 5.7 6.1 Sul 40:0(2OH) 0.71 0.88 1.7E−05 3.4E−02 4.3 1.8 Sul 42:1 (OH) 0.58 0.61 8.5E−061.2E−07 4.9 5.5 Sul 42:1 (2OH) 0.55 0.60 8.8E−04 1.1E−04 3.3 3.8 Sul42:0 (2OH) 0.72 0.85 8.2E−05 2.2E−02 3.9 2.0 SulfoHex₂Cer 42:2 1.34 1.849.1E−02 2.7E−02 −1.4 −2.0 PI 36:4 0.59 0.82 2.7E−05 4.9E−02 4.3 1.7 PI36:3 0.54 0.75 8.6E−06 1.3E−02 4.6 2.3 PI 36:2 0.62 0.81 6.5E−06 1.2E−024.7 2.3 PI 36:1 0.53 0.70 4.7E−07 5.0E−04 5.4 3.4 PI 38:5 0.52 0.747.1E−07 1.1E−02 5.3 2.3 PI 38:3 0.60 0.72 6.6E−06 7.3E−04 4.8 3.3

TABLE 26 Statistical parameters for MALDI-MS analysis of non- cancerousplasma samples and cancerous plasma samples of males (M) and females (F)for prostate cancer. Species Fold change P-value T-value cholesterolsulfate 0.61 7.6E−09 6.1 SM 32:1 0.60 8.9E−12 7.4 SM 33:1 0.59 2.7E−117.2 SM 34:2 0.60 1.6E−10 6.8 SM 34:1 0.65 2.9E−10 6.7 SM 36:2 0.611.2E−08 6.0 SM 36:1 0.66 1.8E−08 5.9 SM 37:1 0.59 3.6E−10 6.7 SM 38:20.58 1.9E−10 6.8 SM 38:1 0.62 2.9E−11 7.2 SM 39:1 0.54 2.5E−13 8.0 SM40:3 0.46 6.2E−09 6.1 SM 40:2 0.58 5.4E−12 7.5 SM 40:1 0.59 6.8E−13 7.9SM 41:2 0.57 1.5E−12 7.7 SM 41:1 0.58 2.1E−13 8.1 SM 42:2 0.64 2.9E−096.3 SM 42:1 0.59 1.6E−12 7.7 SM 43:2 0.56 1.5E−08 5.9 SM 43:1 0.464.0E−08 5.7 Sul 34:1 0.62 4.0E−10 6.7 Sul 34:2 (OH) 0.52 1.9E−08 5.9 Sul34:1 (OH) 0.61 2.4E−09 6.3 Sul 34:0 (OH) 0.45 3.7E−10 6.7 Sul 40:2 0.471.5E−06 5.0 Sul 40:1 0.47 4.0E−12 7.5 Sul 40:2 (OH) 0.49 9.0E−07 5.0 Sul41:1 0.51 2.6E−08 5.8 Sul 40:1 (OH) 0.56 2.6E−11 7.2 Sul 42:2 0.571.8E−08 5.9 Sul 42:1 0.51 2.1E−09 6.3 Sul 41:1 (OH) 0.53 6.7E−11 7.0 Sul42:2 (OH) 0.64 3.8E−08 5.7 Sul 42:1 (OH) 0.59 3.5E−11 7.1 Sul 42:1 (2OH)0.55 1.0E−06 5.0 PI 36:4 0.59 1.5E−06 4.9 PI 36:3 0.57 3.0E−06 4.7 PI38:5 0.53 1.1E−07 5.5 PI 38:4 0.60 1.8E−09 6.3 PI 38:3 0.61 7.8E−09 6.0

TABLE 27 Statistical parameters for the MALDI-MS analysis ofnon-cancerous urine samples and cancerous urine samples of males (M) andfemales (F) for kidney and prostate cancer. Kidney Prostate Fold Foldchange P-value T-value change P-value T-value Species F M F M F M M M Mcortisol sulfate 1.9 3.8 5.2E−02 1.3E−03 −1.7 −3.1 1.5 5.2E−02 −1.7lithocholic acid sulfate 1.4 2.4 2.0E−01 4.4E−02 −0.9 −1.7 1.1 3.8E−01−0.3 cholesterol sulfate 1.1 1.7 3.8E−01 1.3E−02 −0.3 −2.3 1.5 1.0E−01−1.3 taurolithocholic acid 0.6 1.6 1.0E−01 9.7E−02 1.3 −1.3 1.1 3.9E−01−0.3 sulfocholic acid 2.3 6.6 5.7E−02 5.7E−03 −1.7 −2.6 1.9 1.7E−02 −2.2taurodeoxycholic acid 0.8 2.8 3.3E−01 8.6E−03 0.5 −2.4 1.5 8.9E−02 −1.4sulfoglycolithocholic acid 1.0 1.6 4.4E−01 2.9E−02 0.2 −1.9 1.2 2.4E−01−0.7 taurocholic acid 0.6 2.0 8.6E−02 1.3E−02 1.4 −2.3 1.6 8.6E−02 −1.4glycochenodeoxycholic acid 1.0 2.3 4.8E−01 1.5E−02 0.0 −2.2 1.2 2.6E−01−0.7 sulfate psychosine sulfate 1.1 1.4 3.8E−01 5.2E−02 −0.3 −1.7 1.12.8E−01 −0.6 Sul 34:1 (OH) 1.1 1.0 3.8E−01 4.4E−01 −0.3 −0.1 1.0 4.3E−010.2 Sul 36:1 (OH) 1.7 1.8 7.8E−02 3.9E−03 −1.5 −2.8 1.3 1.2E−01 −1.2 Sul38:1 1.2 1.0 3.2E−01 4.9E−01 −0.5 0.0 0.8 3.0E−01 0.5 Sul 38:1 (OH) 1.10.7 4.3E−01 1.3E−01 −0.2 1.1 0.8 1.9E−01 0.9 Sul 40:2 1.6 1.5 1.8E−018.2E−02 −1.0 −1.4 0.8 2.1E−01 0.8 Sul 41:2 1.8 1.9 7.9E−02 1.8E−03 −1.5−3.0 1.4 9.3E−02 −1.3 Sul 41:1 0.9 1.0 4.1E−01 4.5E−01 0.2 0.1 0.93.2E−01 0.5 Sul 40:1 (OH) 0.8 0.9 3.0E−01 3.4E−01 0.5 0.4 0.9 2.6E−010.7 Sul 40:0 (OH) 0.7 1.0 1.6E−01 4.5E−01 1.0 0.1 0.9 2.9E−01 0.6 Sul42:3 2.9 3.6 3.8E−02 3.3E−04 −1.9 −3.6 1.6 2.4E−02 −2.0 Sul 42:2 2.2 2.15.7E−02 5.1E−03 −1.7 −2.6 1.0 4.4E−01 −0.1 Sul 42:1 1.2 1.4 2.8E−018.3E−02 −0.6 −1.4 1.0 4.4E−01 −0.1 Sul 41:1 (OH) 0.9 0.9 3.6E−01 3.6E−010.4 0.4 0.9 2.8E−01 0.6 Sul 41:0 (OH) 0.8 0.9 2.3E−01 3.3E−01 0.8 0.50.8 2.4E−01 0.7 Sul 40:0 (2OH) 0.7 0.9 1.7E−01 3.3E−01 1.0 0.5 0.82.1E−01 0.8 Sul 42:3 (OH) 1.6 2.0 8.5E−02 6.7E−04 −1.4 −3.3 1.5 5.6E−02−1.6 Sul 41:0 (2OH) 0.8 0.9 2.0E−01 3.9E−01 0.9 0.3 0.9 2.6E−01 0.6 Sul43:2 (OH) 1.5 1.9 1.2E−01 1.0E−03 −1.2 −3.2 1.4 7.1E−02 −1.5 Sul 42:1(2OH) 0.8 1.1 2.0E−01 2.8E−01 0.9 −0.6 0.9 3.2E−01 0.5 Sul 42:0 (2OH)0.8 1.0 2.8E−01 4.5E−01 0.6 −0.1 0.9 3.7E−01 0.3 Sul 43:1 (2OH) 1.7 2.17.7E−02 3.7E−04 −1.5 −3.5 1.6 3.3E−02 −1.9 Sul 43:0 (2OH) 1.4 1.31.8E−01 8.7E−02 −0.9 −1.4 1.0 5.0E−01 0.0 SulfoHex₂Cer 34:1 2.9 2.81.9E−02 2.6E−03 −2.2 −2.9 1.4 1.1E−01 −1.3 SulfoHex₂Cer 34:1 (OH) 2.11.8 5.4E−02 6.3E−03 −1.7 −2.6 1.6 7.2E−02 −1.5 SulfoHex₂Cer 36:1 2.2 3.44.1E−02 1.8E−04 −1.8 −3.7 1.7 1.7E−02 −2.2 SulfoHex₂Cer 38:1 3.1 3.44.2E−02 2.1E−03 −1.8 −3.0 1.6 5.4E−02 −1.6 SulfoHex₂Cer 38:1 (OH) 1.91.9 4.9E−02 5.5E−03 −1.7 −2.6 1.5 8.7E−02 −1.4 SulfoHex₂Cer 40:2 3.0 3.83.7E−02 1.2E−03 −1.9 −3.2 1.6 3.2E−02 −1.9 SulfoHex₂Cer 40:1 3.7 2.53.5E−02 2.4E−03 −1.9 −2.9 1.3 1.7E−01 −1.0 SulfoHex₂Cer 41:1 2.3 2.74.3E−02 6.3E−05 −1.8 −4.0 1.6 2.8E−02 −2.0 SulfoHex₂Cer 40:1 (OH) 1.70.8 1.8E−01 2.0E−01 −1.0 0.9 0.7 1.3E−01 1.2 SulfoHex₂Cer 42:3 2.6 3.53.9E−02 1.3E−03 −1.9 −3.1 1.6 2.7E−02 −2.0 SulfoHex₂Cer 42:2 4.5 3.32.5E−02 1.1E−03 −2.1 −3.2 1.2 2.4E−01 −0.7 SulfoHex₂Cer 42:1 3.3 2.63.5E−02 1.1E−03 −1.9 −3.2 1.3 1.6E−01 −1.0 SulfoHex₂Cer 41:1 (OH) 1.81.7 7.9E−02 1.0E−02 −1.5 −2.4 1.3 1.6E−01 −1.0 SulfoHex₂Cer 40:0 (2OH)1.2 0.8 3.1E−01 2.2E−01 −0.5 0.8 0.7 7.4E−02 1.5

EXAMPLE C: UHPSFC/MS LIPIDOMIC ANALYSIS OF HUMAN PLASMA OF CANCERPATIENTS FOR THE DIFFERENTIATION OF VARIOUS CANCEROUS SAMPLES

The aim of the invention is not only to differentiate non-cancerous andcancerous samples from body fluids, but also the prediction of thelocalization of the cancer in the body. It was found that there is adifference of the lipid profile depending on the cancer type, which canbe determined in the biological fluid. OPLS-DA could be again a tool tovisualize the most probably minor differences of the lipid profile.

FIG. 19 represents the OPLS-DA plot of various cancer type samples(breast, kidney, prostate) from males. This figure confirms that thereis a difference in the lipid profile of different cancer types.

FIG. 20 shows the OPLS-DA plot for the differentiation of prostate andkidney cancer samples from males. The sensitivity is 76.1% and thespecificity is 80.3% for males.

FIG. 21 represents the OPLS-DA plot for the differentiation of breastand kidney cancer samples from females. The sensitivity is 74.2% and thespecificity is 96.8% for females.

EXAMPLE D—EXPERIMENTAL RESULTS RELATING TO DIAGNOSTICS OF PANCREATICCANCER

All quantified lipids in human serum of males (M) and females (F) forpancreatic cancer patients and healthy volunteers are summarized inTable 28 for shotgun measurements, in Table 29 for UHPSFC/MSmeasurements and in Table 30 for MALDI-MS measurements, together withfold changes, p-values, and T-values.

Pancreatic Cancer Model Based on Known Classification Using Shotgun andUHPSFC/MS Data:

These statistical models are built based on 292 known serum samples bothfor shotgun and UHPSFC/MS data sets. Absolute concentrations(normalization to internal standard) are used for these statisticalanalyses. First, the non-supervised PCA method is used to check theregular distribution of data, and it already shows partial separation ofclasses of cancer patients and healthy volunteers (controls). Then,supervised OPLS-DA model is prepared to improve class separation ofnon-cancerous and cancerous serum samples (FIG. 22). For this example ofcombination of data obtained using UHPSFC/MS and shotgun MS,specificitity 98.7% and sensitivity 98.6% (accuracy 98.6%) is obtained.These models include all lipids used (complete lipidome) and areautomatically computed by SIMCA software. FIGS. 23 (males) and FIG. 24(females) show subsequently the OPLS-DA statistical models after genderseparation. These models again illustrate another improvement ofnon-cancerous and cancerous samples differentiation, where sensitivity100%, specificity 100% (accuracy 100%) is obtained in case of males andsensitivity 100%, specificity 95.9% (accuracy 98.7%) is obtained forfemales. It is worth noting that there is the gap between both groups,which visually highlights the reliability of the model for pancreaticcancer classification.

Pancreatic Cancer Model Based on Unknown Classification Using Shotgunand UHPSFC/MS Data:

The next step is the verification of the quality of OPLS-DA modelprepared for samples with known classification for blinded samples withunknown classification. The probability of being the cancer patient isestimated using 4 final models: #1/OPLS-DA of shotgun MS, #2/OPLS-DA ofUHPSFC/MS, #3/OPLS-DA of all data, #4 Support vector machines (SVM) ofall data, and finally the average of all models, which is used for thefinal assignment. The observation is classified as tumor if thisestimate is bigger than 0.5 and as healthy volunteer if this probabilityis smaller than 0.5 (FIGS. 25 and 26). Triangle symbols showsobservations “sure healthy”, square symbols as “healthy”, star symbolsas “cancer” and diamond symbols as “sure cancer”. The models resulted invery good prediction ability as was expected (4 wrongly classified from73 unknown samples). Tables 31 and 32 show the resulting assignments ofsamples.

TABLE 28 List of all quantified lipids with shotgun measurement togetherwith fold changes, p-values, and T-values. Fold change p-value T-valueLipid M F M F M F CE 14:0 0.88 0.73 9.7E−02 1.7E−04 1.3 3.8 CE 16:0 1.020.96 3.5E−01 2.4E−01 −0.4 0.7 CE 16:1 1.35 0.83 2.7E−03 1.8E−02 −2.8 2.1CE 18:1 1.15 0.94 1.2E−02 1.9E−01 −2.3 0.9 CE 18:2 0.90 0.80 2.9E−025.2E−04 2.0 3.4 CE 18:3 0.87 0.70 4.6E−02 1.3E−04 1.7 3.9 CE 20:4 0.941.00 1.9E−01 4.8E−01 0.9 0.1 CE 20:5 0.65 0.77 1.0E−02 1.1E−02 2.5 2.4CE 22:4 1.09 0.90 7.5E−02 1.3E−01 −1.5 1.1 CE 22:5 2.68 0.83 1.5E−052.1E−01 −4.4 0.8 CE 22:6 0.95 1.01 2.4E−01 4.6E−01 0.7 −0.1 Cerd18:0/16:1; Cerd18:0/15:2(1OH) 0.92 1.00 5.6E−02 5.0E−01 1.6 0.0 Cerd18:0/16:2 0.96 0.96 2.1E−01 2.1E−01 0.8 0.8 Cer d18:0/18:2 0.96 0.972.6E−01 2.3E−01 0.6 0.8 Cer d18:0/24:0; Cer d18:0/23:1(1OH) 0.94 0.752.9E−01 3.1E−03 0.6 2.8 Cer d18:0/24:1; Cer d18:0/23:2(1OH) 1.13 1.001.8E−02 4.6E−01 −2.2 −0.1 Cer d18:1/16:0; Cer d18:1/15:1(1OH) 1.64 1.291.8E−12 2.6E−03 −8.1 −2.9 Cer d18:1/16:1; Cer d18:1/15:2(1OH) 1.00 0.924.8E−01 4.9E−02 −0.1 1.7 Cer d18:1/16:2 1.08 0.89 9.4E−02 1.4E−02 −1.42.3 Cer d18:1/18:0; Cer d18:1/17:1(1OH) 1.71 1.54 4.3E−13 6.2E−06 −8.1−4.5 Cer d18:1/18:2 1.07 0.89 1.2E−01 1.1E−02 −1.2 2.4 Cer d18:1/18:31.21 0.79 1.0E−03 1.7E−04 −3.2 3.8 Cer d18:1/20:0; Cer d18:1/19:1(1OH)1.23 1.28 5.9E−02 2.1E−02 −1.6 −2.1 Cer d18:1/22:0; Cer d18:1/21:1(1OH)1.10 1.09 6.8E−02 1.0E−01 −1.5 −1.3 Cer d18:1/23:0; Cer d18:1/22:1(1OH)0.85 0.77 1.5E−02 3.0E−04 2.3 3.6 Cer d18:1/24:0; Cer d18:1/23:1(1OH)0.87 0.78 1.8E−02 4.9E−04 2.2 3.4 Cer d18:1/24:1; Cer d18:1/23:2(1OH)1.53 1.36 6.2E−13 4.8E−05 −8.3 −4.1 Cer d18:1/25:0; Cer d18:1/24:1(1OH)0.95 0.73 1.9E−01 2.5E−05 0.9 4.3 DG 30:0 1.05 1.10 3.6E−01 1.7E−01 −0.4−1.0 DG 31:0 1.11 0.95 7.3E−02 2.5E−01 −1.5 0.7 DG 32:0 1.14 1.351.4E−01 3.4E−03 −1.1 −2.8 DG 32:1 1.10 1.12 2.5E−01 1.2E−01 −0.7 −1.2 DG32:2 0.98 1.04 4.2E−01 3.1E−01 0.2 −0.5 DG 34:0 0.59 1.06 1.1E−032.1E−01 3.4 −0.8 DG 34:1 1.11 1.27 2.1E−01 1.1E−02 −0.8 −2.3 DG 34:21.05 1.13 3.4E−01 9.1E−02 −0.4 −1.4 DG 34:3 0.91 0.98 2.1E−01 4.3E−010.8 0.2 DG 36:0 0.35 0.88 2.7E−03 1.0E−01 3.1 1.3 DG 36:1 1.01 1.334.6E−01 2.1E−03 −0.1 −2.9 DG 36:2 1.09 1.35 2.6E−01 1.1E−03 −0.7 −3.2 DG36:3 0.89 1.23 1.6E−01 1.7E−02 1.0 −2.2 DG 36:4 0.78 1.10 1.8E−021.8E−01 2.2 −0.9 DG 38:2 1.07 1.03 2.2E−01 3.3E−01 −0.8 −0.4 DG 38:31.09 0.90 1.9E−01 9.9E−02 −0.9 1.3 DG 38:4 1.03 1.06 3.8E−01 2.3E−01−0.3 −0.7 DG 38:5 0.92 1.24 2.2E−01 2.2E−03 0.8 −2.9 DG 38:6 0.77 1.191.8E−02 8.9E−02 2.2 −1.4 Hex2Cer d18:1/16:0; Hex2Cer d18:1/15:1(1OH)1.25 1.08 8.2E−05 1.5E−01 −3.9 −1.1 Hex2Cer d18:1/20:0;Hex2Cerd18:1/19:1(1OH) 1.32 0.83 8.0E−02 1.3E−01 −1.4 1.2 Hex2Cerd18:1/21:0(1OH) 1.12 0.95 1.8E−02 2.3E−01 −2.1 0.8 Hex2Cer d18:1/22:0;Hex2Cer d18:1/21:1(1OH) 0.95 0.80 2.1E−01 1.0E−03 0.8 3.2 Hex2Cerd18:1/24:1; Hex2Cer d18:1/23:2(1OH) 1.34 1.19 6.6E−05 2.2E−02 −4.1 −2.1HexCer d18:1/16:0; HexCer d18:1/15:1(1OH) 1.50 1.12 1.9E−13 7.8E−02 −8.3−1.4 HexCer d18:1/20:0; HexCer d18:1/19:1(1OH) 1.19 1.10 3.3E−02 2.1E−01−1.9 −0.8 HexCer d18:1/22:0; HexCer d18:1/21:1(1OH) 1.02 0.87 3.4E−011.7E−02 −0.4 2.2 HexCer d18:1/24:0; HexCer d18:1/23:1(1OH) 0.96 0.792.6E−01 7.7E−04 0.7 3.3 HexCer d18:1/24:1; HexCer d18:1/23:2(1OH) 1.561.14 8.7E−13 5.1E−02 −7.9 −1.7 HexCer d18:1/25:0; HexCer d18:1/24:1(1OH)0.91 1.10 2.2E−02 1.1E−02 2.1 −2.3 Chol 1.12 0.95 8.3E−03 2.8E−01 −2.50.6 LPC 16:0 0.86 0.82 3.1E−03 1.1E−03 2.9 3.2 LPC 16:1 0.97 0.753.5E−01 2.1E−04 0.4 3.7 LPC 18:0 0.77 0.80 1.5E−04 1.9E−03 3.9 3.0 LPC18:1 0.92 0.85 8.7E−02 8.3E−03 1.4 2.5 LPC 18:2 0.62 0.68 3.4E−073.0E−07 5.8 5.4 LPC 20:1 1.13 0.74 1.7E−01 6.1E−04 −1.0 3.4 LPC 20:30.80 0.76 8.4E−04 5.8E−05 3.3 4.1 LPC 20:4 0.75 0.87 2.0E−05 2.3E−02 4.52.0 LPC 22:6 0.72 1.00 1.7E−06 4.8E−01 5.2 0.1 LPE 16:0 1.12 0.941.2E−01 2.1E−01 −1.2 0.8 LPE 18:0 1.02 0.89 4.3E−01 6.9E−02 −0.2 1.5 LPE18:1 0.92 0.84 2.5E−01 3.5E−02 0.7 1.8 LPE 18:2 0.79 0.83 4.7E−022.2E−02 1.7 2.1 LPE 20:4 0.78 1.25 2.1E−02 3.7E−02 2.1 −1.8 LPE 22:60.98 1.16 4.4E−01 6.2E−02 0.2 −1.6 LPG 20:0 0.87 0.89 2.8E−01 3.0E−010.6 0.5 LPG O-18:0; LPG 17:0 0.85 1.16 2.0E−01 1.7E−01 0.8 −1.0 LPGO-20:0; LPG 19:0 0.93 1.21 3.3E−01 9.6E−02 0.4 −1.3 LPG O-22:0; LPG 21:00.91 1.32 2.4E−01 1.6E−02 0.7 −2.2 MG 16:0 0.89 1.13 4.3E−02 4.6E−02 1.8−1.7 MG 17:0 0.67 1.54 5.6E−02 1.2E−02 1.7 −2.3 MG 18:0 0.67 1.072.3E−03 1.3E−01 3.1 −1.1 MG 18:1 1.35 1.72 3.3E−02 8.8E−03 −1.9 −2.4 MG22:0 1.19 1.04 1.3E−01 4.0E−01 −1.1 −0.3 PA 38:4; PA P-40:10; PA P-29:30.79 2.08 5.4E−03 7.7E−07 2.7 −5.1 PC 30:0 1.21 0.92 6.8E−03 1.2E−01−2.6 1.2 PC 32:0; PC O-33:0 1.55 1.10 2.5E−13 1.1E−01 −8.1 −1.2 PC 32:1;PC P-33:0 1.90 0.93 6.1E−06 2.7E−01 −4.6 0.6 PC 32:2; PC P-33:1 0.670.53 1.4E−03 1.6E−06 3.1 5.0 PC 34:1; PC P-35:0 1.46 1.02 1.2E−094.0E−01 −6.5 −0.3 PC 34:2; PC P-35:1 1.10 0.86 2.4E−02 1.1E−02 −2.0 2.4PC 34:3; PC P-35:2 1.06 0.76 1.9E−01 1.2E−04 −0.9 3.9 PC 34:4; PC P-35:30.79 0.70 2.7E−03 2.0E−05 2.9 4.4 PC 36:0; PC P-38:6 0.43 0.50 3.2E−034.7E−04 2.9 3.5 PC 36:1; PC P-37:0 1.30 0.95 2.4E−04 2.6E−01 −3.6 0.7 PC36:2; PC P-37:1 1.02 0.86 3.3E−01 1.8E−02 −0.4 2.2 PC 36:3; PC P-37:21.16 0.85 3.1E−03 9.4E−03 −2.8 2.4 PC 36:4; PC P-37:3 1.04 0.95 2.4E−012.5E−01 −0.7 0.7 PC 36:5; PC P-37:4 0.72 0.81 2.5E−02 1.4E−02 2.1 2.3 PC38:2; PC P-40:8; PC P-39:1 1.10 0.47 3.7E−01 7.9E−04 −0.3 3.3 PC 38:3;PC P-40:9; PC P-39:2 1.12 0.94 2.2E−02 2.1E−01 −2.1 0.8 PC 38:4; PCP-40:10; PC P-29:3 1.00 1.01 4.8E−01 4.6E−01 0.0 −0.1 PC 38:5; PC P-39:40.93 0.92 1.8E−01 1.2E−01 0.9 1.2 PC 38:6; PC P-39:5 0.94 1.02 1.9E−013.8E−01 0.9 −0.3 PC 40:4; PC P-42:10; PC P-41:3 1.17 0.89 7.4E−036.6E−02 −2.5 1.5 PC 40:5; PC P-41:4 1.06 1.03 2.4E−01 3.5E−01 −0.7 −0.4PC 40:6; PC P-41:5 0.98 1.17 4.1E−01 2.9E−02 0.2 −1.9 PC O-32:0; PC 31:01.36 0.91 3.6E−07 1.2E−01 −5.4 1.2 PC O-34:0; PC 33:0 1.16 0.92 3.8E−021.4E−01 −1.8 1.1 PC P-32:0; PC 31:1 1.43 0.82 6.2E−11 2.3E−03 −7.1 3.0PC P-34:0; PC 33:1 1.44 0.96 1.6E−11 3.0E−01 −7.5 0.5 PC P-34:1; PC 33:21.04 0.71 2.7E−01 2.7E−06 −0.6 4.9 PC P-36:0; PC 35:1 1.05 0.71 3.0E−012.2E−04 −0.5 3.7 PC P-36:1; PC 35:2 1.04 0.79 2.8E−01 6.4E−04 −0.6 3.4PC P-36:2; PC 35:3 1.07 0.74 1.3E−01 3.8E−05 −1.1 4.2 PC P-36:3; PC 35:41.06 0.83 1.4E−01 6.8E−03 −1.1 2.5 PC P-36:4; PC 35:5 0.99 0.84 4.5E−017.9E−03 0.1 2.5 PC P-38:2; PC 37:3 0.91 0.69 1.8E−01 5.6E−05 0.9 4.1 PCP-38:3; PC 37:4 1.08 0.87 5.8E−02 2.4E−02 −1.6 2.0 PC P-38:4; PC 37:51.13 0.96 4.1E−03 2.7E−01 −2.7 0.6 PC P-38:5; PC 37:6 0.96 0.88 2.3E−013.7E−02 0.8 1.8 PC P-40:3; PC 39:4 0.96 0.72 2.8E−01 7.2E−05 0.6 4.0 PCP-40:4; PC 39:5 1.01 0.81 4.6E−01 6.5E−03 −0.1 2.6 PC P-40:5; PC 39:61.00 0.97 4.8E−01 3.1E−01 0.0 0.5 PE 32:0; PE O-33:0 1.47 1.27 2.0E−034.4E−02 −3.0 −1.7 PE 32:1; PE P-33:0 2.48 1.19 1.4E−06 6.8E−02 −4.9 −1.5PE 34:0; PE P-36:6 1.07 0.76 2.8E−01 3.2E−03 −0.6 2.8 PE 34:1; PE P-35:01.98 1.20 1.5E−09 4.4E−02 −6.4 −1.7 PE 34:2; PE P-35:1 1.78 1.11 1.2E−071.4E−01 −5.5 −1.1 PE 34:3; PE P-35:2 1.55 0.96 1.9E−05 3.1E−01 −4.3 0.5PE 36:0; PE P-38:6 0.77 0.84 1.5E−03 1.7E−02 3.2 2.2 PE 36:1; PE P-37:01.18 1.06 8.1E−02 2.8E−01 −1.4 −0.6 PE 36:2; PE P-37:1 1.33 1.04 4.8E−033.4E−01 −2.7 −0.4 PE 36:3; PE P-37:2 1.43 0.97 3.1E−03 3.7E−01 −2.8 0.3PE 36:4; PE P-37:3 1.70 1.15 2.1E−08 5.7E−02 −5.9 −1.6 PE 36:5; PEP-37:4 1.26 0.93 1.8E−02 1.9E−01 −2.2 0.9 PE 38:0; PE P-40:6, PE O-39:00.77 0.81 1.8E−04 6.2E−03 3.8 2.6 PE 38:1; PE P-40:7; PE P-39:0 0.940.83 1.5E−01 5.0E−03 1.1 2.7 PE 38:2; PE P-40:8; PE P-39:1 1.05 1.512.4E−01 6.9E−04 −0.7 −3.3 PE 38:3; PE P-40:9; PE P-39:2 1.67 0.904.6E−05 1.6E−01 −4.1 1.0 PE 38:4; PE P-40:10; PE P-29:3 1.41 1.143.8E−05 6.0E−02 −4.2 −1.6 PE 38:5; PE P-39:4 1.49 1.02 2.3E−05 3.9E−01−4.3 −0.3 PE 38:6; PE P-39:5 1.65 1.19 1.3E−07 4.4E−02 −5.6 −1.7 PE38:7; PE O-38:0; PE 37:0 1.36 0.96 9.7E−06 3.0E−01 −4.5 0.5 PE 40:1; PEP-42:7; PE P-41:0 0.88 1.05 2.2E−01 3.7E−01 0.8 −0.3 PE 40:2; PE P-42:8;PE P-41:1 0.92 0.94 2.6E−01 3.4E−01 0.6 0.4 PE 40:3; PE P-42:9; PEP-41:2 0.85 0.89 6.9E−02 1.7E−01 1.5 1.0 PE 40:4; PE P-42:10; PE P-41:31.50 1.03 9.7E−05 3.3E−01 −3.9 −0.4 PE 40:5; PE P-41:4 1.54 1.13 3.9E−059.2E−02 −4.1 −1.3 PE 40:6; PE P-41:5 1.42 1.30 2.8E−04 3.3E−03 −3.6 −2.8PE 40:7; PE O-40:0; PE 39:0 1.51 1.03 1.9E−06 3.6E−01 −4.9 −0.4 PE 40:8;PE P-40:0; PE 39:1 1.19 0.96 3.9E−03 3.0E−01 −2.7 0.5 PE 42:9; PEP-42:1; PE 41:2 0.97 1.09 4.1E−01 3.0E−01 0.2 −0.5 PE P-34:0; PE 33:11.03 0.66 3.5E−01 9.1E−08 −0.4 5.8 PE P-34:1; PE 33:2 0.86 0.67 6.1E−026.0E−06 1.6 4.7 PE P-36:0; PE 35:1 1.31 0.89 4.1E−02 2.2E−01 −1.8 0.8 PEP-36:1; PE 35:2 1.12 0.80 6.7E−02 9.5E−04 −1.5 3.2 PE P-36:2; PE 35:30.89 0.69 8.8E−02 7.9E−06 1.4 4.6 PE P-36:3; PE 35:4 0.72 0.56 8.8E−042.6E−06 3.3 5.0 PE P-36:4; PE 35:5 0.76 0.79 5.3E−04 4.0E−03 3.5 2.7 PEP-38:2; PE 37:3 1.39 0.57 4.9E−02 5.1E−06 −1.7 4.8 PE P-38:3; PE 37:40.87 0.69 4.8E−02 8.8E−05 1.7 4.0 PE P-38:4; PE 37:5 0.74 0.68 1.9E−041.7E−05 3.8 4.4 PE P-38:5; PE 37:6 0.73 0.71 3.6E−05 5.7E−05 4.3 4.1 PEP-40:4; PE 39:5 0.91 0.80 9.6E−02 8.9E−04 1.3 3.3 PE P-40:5; PE 39:60.83 0.78 8.9E−03 8.0E−04 2.5 3.3 PG 32:0; PG O-33:0 0.84 1.35 2.6E−016.7E−02 0.7 −1.5 PG 32:1; PG P-33:0 0.90 1.32 3.6E−01 6.9E−02 0.4 −1.5PG 34:0; PG P-36:6 0.61 0.96 5.7E−02 4.2E−01 1.6 0.2 PG 34:1; PG P-35:00.86 1.24 2.5E−01 6.5E−02 0.7 −1.5 PG 34:2; PG P-35:1 0.76 1.09 1.1E−012.8E−01 1.3 −0.6 PG 36:1; PG P-37:0 0.86 1.13 1.7E−01 1.5E−01 1.0 −1.0PG 36:2; PG P-37:1 0.83 1.14 1.8E−01 1.4E−01 1.0 −1.1 PG 36:3; PG P-37:20.64 0.67 1.7E−01 2.6E−02 1.0 2.0 PG 36:4; PG P-37:3 0.70 1.19 5.1E−021.8E−01 1.7 −0.9 PI 32:0; PI O-33:0 0.90 1.27 3.1E−01 1.2E−01 0.5 −1.2PI 32:1; PI P-33:0 1.63 0.92 3.4E−03 2.7E−01 −2.8 0.6 PI 34:0; PI P-36:60.92 0.81 4.0E−01 1.5E−01 0.3 1.0 PI 34:1; PI P-35:0 1.58 0.86 9.7E−066.3E−02 −4.5 1.6 PI 34:2; PI P-35:1 1.53 0.98 2.3E−06 4.1E−01 −4.8 0.2PI 36:0; PI P-38:6 1.21 0.82 1.8E−01 1.7E−01 −0.9 0.9 PI 36:1; PI P-37:01.34 0.75 4.4E−03 2.8E−03 −2.7 2.9 PI 36:2; PI P-37:1 1.38 0.92 2.7E−041.7E−01 −3.6 1.0 PI 36:3; PI P-37:2 1.53 0.95 1.2E−05 2.9E−01 −4.5 0.6PI 36:4; PI P-37:3 1.34 0.95 7.3E−04 3.1E−01 −3.3 0.5 PI 38:2; PIP-40:8; PI P-39:1 1.43 0.90 2.0E−04 1.4E−01 −3.7 1.1 PI 38:3; PI P-40:9;PI P-39:2 1.26 0.80 2.7E−02 2.8E−02 −2.0 1.9 PI 38:4; PI P-40:10; PIP-29:3 1.21 0.98 2.3E−02 4.0E−01 −2.1 0.3 PI 38:5; PI P-39:4 1.34 0.921.0E−03 2.0E−01 −3.2 0.8 PI 38:6; PI P-39:5 1.54 1.02 4.2E−04 4.4E−01−3.5 −0.2 PI 40:0; PI P-42:6, PI O-41:0 1.16 0.95 1.3E−01 2.9E−01 −1.10.6 PI 40:4; PI P-42:10; PI P-41:3 1.52 0.98 2.2E−04 4.4E−01 −3.7 0.2 PI40:5; PI P-41:4 1.44 1.05 1.2E−04 2.9E−01 −3.9 −0.6 PI 40:6; PI P-41:51.71 1.09 1.8E−06 2.1E−01 −4.9 −0.8 PI 42:3; PI P-43:2 1.32 0.93 1.6E−022.8E−01 −2.2 0.6 PI P-36:0; PI 35:1 1.30 0.89 1.0E−01 2.9E−01 −1.3 0.6PI P-36:1; PI 35:2 1.07 1.01 3.4E−01 4.8E−01 −0.4 −0.1 PI P-38:3; PI37:4 1.22 0.92 4.3E−02 2.7E−01 −1.8 0.6 PS 34:0; PS P-36:6 1.36 1.125.4E−03 2.2E−01 −2.6 −0.8 PS 34:2; PS P-35:1 1.03 1.13 3.4E−01 1.1E−01−0.4 −1.2 PS 36:1; PS P-37:0 1.46 1.07 1.1E−02 3.3E−01 −2.4 −0.5 PS38:4; PS P-40:10; PS P-29:3 1.29 1.48 7.9E−03 2.1E−04 −2.5 −3.6 PS 38:6;PS P-39:5 0.88 1.40 7.0E−02 3.9E−03 1.5 −2.7 PS 42:1; PS P-43:0 0.821.34 8.1E−02 2.0E−02 1.4 −2.1 PS P-32:0; PS 31:1 1.23 0.98 7.5E−044.3E−01 −3.3 0.2 SM 32:1 0.93 0.84 1.1E−01 7.9E−03 1.2 2.5 SM 33:1 1.030.94 3.0E−01 2.2E−01 −0.5 0.8 SM 34:0 1.38 1.09 2.6E−06 1.1E−01 −4.8−1.2 SM 34:1 1.28 1.06 2.7E−09 1.6E−01 −6.5 −1.0 SM 34:2 1.07 0.976.2E−02 3.1E−01 −1.6 0.5 SM 35:1 0.77 1.65 1.3E−03 1.2E−03 3.1 −3.2 SM36:0 2.02 1.94 5.5E−03 3.0E−03 −2.6 −2.8 SM 36:1 1.16 1.28 1.7E−021.3E−03 −2.2 −3.1 SM 36:2 1.10 1.11 3.0E−02 6.5E−02 −1.9 −1.5 SM 37:00.83 0.76 2.6E−01 1.5E−01 0.7 1.1 SM 39:1 0.71 0.65 1.9E−05 2.4E−05 4.64.4 SM 40:0 0.96 0.84 2.8E−01 1.4E−02 0.6 2.3 SM 40:1 0.90 0.84 6.0E−022.1E−02 1.6 2.1 SM 41:1 0.78 0.70 6.5E−05 5.6E−06 4.2 4.7 SM 41:2 0.910.80 4.8E−02 1.5E−03 1.7 3.1 SM 42:2 1.31 1.10 1.1E−09 9.3E−02 −6.6 −1.3SM 43:1 1.45 0.60 3.8E−02 1.6E−05 −1.8 4.4 SM 44:2 1.65 0.93 6.2E−032.6E−01 −2.6 0.6 SulfoHexCer 41:0; SulfoHexCer 40:1(1OH) 1.06 1.142.1E−01 7.7E−02 −0.8 −1.5 SulfoHexCer 41:1; SulfoHexCer 40:2(1OH) 0.920.83 1.4E−01 3.3E−02 1.1 1.9 SulfoHexCer 43:0; SulfoHexCer 42:1(1OH)1.20 0.86 2.7E−02 6.5E−02 −2.0 1.5 SulfoHexCer 43:1; SulfoHexCer42:2(1OH) 0.72 1.07 2.7E−04 2.9E−01 3.7 −0.5 SulfoHexCer 43:2;SulfoHexCer 42:3(1OH) 1.45 0.98 7.8E−03 4.6E−01 −2.5 0.1 TG 56:3 0.801.75 3.1E−02 6.5E−06 1.9 −4.6 TG 56:4 1.02 1.22 4.2E−01 7.5E−03 −0.2−2.5 TG 56:5 1.26 1.26 5.5E−03 3.6E−03 −2.6 −2.7 TG 56:6 0.71 1.813.9E−03 2.0E−04 2.8 −3.7 TG 58:6 0.89 1.55 1.6E−01 2.5E−03 1.0 −2.9

TABLE 29 List of all quantified lipids with UHPSFC/MS measurementtogether with fold changes, p-values, and T-values. Fold change p-valueT-value Lipid M F M F M F CE 16:1 1.52 0.85 2.6E−03 7.2E−02 −2.9 1.5 CE16:0 1.02 1.07 3.9E−01 2.5E−01 −0.3 −0.7 CE 18:3 0.78 0.68 8.2E−032.7E−04 2.5 3.6 CE 18:2 0.93 0.86 3.3E−02 3.5E−03 1.9 2.8 CE 18:1 1.190.98 7.9E−03 4.1E−01 −2.5 0.2 CE 18:0 0.50 0.87 8.3E−05 2.3E−01 4.3 0.7CE 20:5 0.60 0.71 3.5E−02 1.5E−02 1.9 2.2 CE 20:4 0.97 0.97 3.3E−013.8E−01 0.5 0.3 CE 20:3 0.97 0.83 3.9E−01 9.0E−02 0.3 1.4 CE 20:2 0.770.86 3.9E−03 9.1E−02 2.8 1.4 CE 22:6 0.91 1.11 1.7E−01 1.7E−01 1.0 −1.0CE 22:5 1.45 0.88 1.9E−03 2.0E−01 −3.0 0.9 TG 44:2 1.09 1.40 4.3E−011.2E−01 −0.2 −1.2 TG 44:1 1.22 1.39 3.5E−01 2.8E−01 −0.4 −0.6 TG 44:00.34 1.41 2.3E−01 3.7E−01 0.8 −0.3 TG 46:3 0.71 1.31 2.3E−01 1.6E−01 0.8−1.0 TG 46:2 0.94 1.16 4.4E−01 2.5E−01 0.2 −0.7 TG 46:1 1.19 1.203.2E−01 1.9E−01 −0.5 −0.9 TG 46:0 1.27 1.27 2.6E−01 1.7E−01 −0.6 −1.0 TG47:2 0.80 1.16 2.5E−01 2.4E−01 0.7 −0.7 TG 47:1 0.98 1.23 4.8E−011.2E−01 0.1 −1.2 TG 47:0 1.08 1.30 4.2E−01 9.5E−02 −0.2 −1.3 TG 48:40.58 1.17 4.9E−02 2.0E−01 1.7 −0.9 TG 48:3 0.70 1.02 9.8E−02 4.6E−01 1.3−0.1 TG 48:2 1.03 1.02 4.5E−01 4.4E−01 −0.1 −0.1 TG 48:1 1.20 1.112.3E−01 3.0E−01 −0.7 −0.5 TG 48:0 1.29 1.24 1.7E−01 1.7E−01 −1.0 −1.0 TG49:3 0.76 1.15 1.3E−01 1.7E−01 1.2 −1.0 TG 49:2 0.90 1.14 3.3E−011.8E−01 0.5 −0.9 TG 49:1 1.01 1.25 4.9E−01 9.4E−02 0.0 −1.3 TG 50:5 0.521.14 1.2E−02 1.9E−01 2.4 −0.9 TG 50:4 0.66 1.01 1.9E−02 4.8E−01 2.2 0.0TG 50:3 0.89 1.04 2.3E−01 3.5E−01 0.8 −0.4 TG 50:2 1.29 1.18 4.5E−026.5E−02 −1.7 −1.5 TG 50:1 1.42 1.36 1.2E−02 9.7E−03 −2.3 −2.4 TG 51:61.20 1.11 3.9E−02 1.0E−01 −1.8 −1.3 TG 51:5 1.18 1.37 5.4E−02 7.9E−04−1.7 −3.3 TG 51:4 0.65 1.32 9.0E−03 1.2E−02 2.5 −2.3 TG 51:3 0.87 1.212.0E−01 3.2E−02 0.9 −1.9 TG 51:2 1.10 1.29 2.9E−01 1.2E−02 −0.6 −2.3 TG51:1 1.05 1.45 4.2E−01 9.3E−03 −0.2 −2.4 TG 52:7 0.44 1.30 1.6E−029.7E−02 2.3 −1.3 TG 52:6 0.51 1.16 7.3E−03 1.6E−01 2.6 −1.0 TG 52:5 0.651.08 9.1E−03 2.9E−01 2.5 −0.6 TG 52:4 0.83 1.16 7.8E−02 7.4E−02 1.5 −1.5TG 52:3 1.05 1.26 3.0E−01 5.6E−03 −0.5 −2.6 TG 52:2 1.31 1.33 3.9E−039.6E−04 −2.8 −3.2 TG 52:1 1.07 1.52 3.7E−01 9.6E−03 −0.3 −2.4 TG 52:01.00 1.35 5.0E−01 4.1E−03 0.0 −2.7 TG 53:5 0.91 1.46 2.4E−01 1.6E−04 0.7−3.7 TG 53:4 0.79 1.32 8.7E−02 1.1E−02 1.4 −2.3 TG 53:3 0.99 1.344.6E−01 2.2E−03 0.1 −2.9 TG 53:2 1.11 1.41 2.4E−01 4.5E−04 −0.7 −3.4 TG54:8 0.39 1.23 4.3E−03 1.9E−01 2.8 −0.9 TG 54:7 0.54 1.24 7.6E−031.0E−01 2.6 −1.3 TG 54:6 0.72 1.35 2.4E−02 1.4E−02 2.1 −2.2 TG 54:5 0.881.32 1.9E−01 9.4E−03 0.9 −2.4 TG 54:4 0.99 1.34 4.8E−01 5.6E−03 0.0 −2.6TG 54:3 1.23 1.45 8.1E−02 4.2E−04 −1.4 −3.5 TG 54:2 1.23 1.56 9.7E−025.5E−04 −1.3 −3.4 TG 54:1 0.73 1.58 1.3E−01 3.2E−02 1.2 −1.9 TG 54:00.93 1.66 3.0E−01 3.2E−06 0.5 −4.8 TG 55:6 1.15 1.62 1.4E−01 1.5E−06−1.1 −4.9 TG 55:4 0.95 1.61 3.6E−01 5.3E−05 0.4 −4.0 TG 55:3 1.08 1.612.9E−01 8.2E−06 −0.6 −4.5 TG 56:9 0.46 1.35 2.6E−03 5.0E−02 3.0 −1.7 TG56:8 0.62 1.53 1.3E−02 2.4E−03 2.4 −2.9 TG 56:7 0.79 1.52 7.6E−027.0E−04 1.5 −3.3 TG 56:6 1.04 1.51 3.7E−01 2.2E−04 −0.3 −3.6 TG 56:51.38 1.68 3.4E−03 1.3E−05 −2.8 −4.4 TG 56:4 1.14 1.30 1.7E−01 9.7E−03−1.0 −2.4 TG 56:3 1.08 1.39 3.1E−01 7.3E−03 −0.5 −2.5 TG 56:2 0.76 1.191.1E−01 1.4E−01 1.3 −1.1 TG 56:1 0.62 1.38 5.6E−02 1.1E−01 1.6 −1.2 TG58:10 0.58 1.62 1.4E−02 1.4E−03 2.3 −3.1 TG 58:9 0.78 1.64 7.5E−021.5E−04 1.5 −3.7 TG 58:8 0.95 1.72 3.7E−01 2.1E−05 0.3 −4.3 TG 58:7 1.111.37 1.6E−01 7.7E−05 −1.0 −3.9 TG 58:6 1.35 1.79 2.4E−02 7.2E−06 −2.0−4.5 TG 58:5 1.33 1.79 2.8E−02 5.7E−06 −2.0 −4.6 DG 34:2 1.06 1.283.5E−01 4.1E−02 −0.4 −1.8 DG 34:1 1.23 1.23 8.5E−02 8.1E−02 −1.4 −1.4 DG34:0 0.67 2.27 4.6E−03 2.5E−08 2.7 −5.8 DG 36:4 0.65 1.38 7.6E−033.9E−02 2.6 −1.8 DG 36:3 0.89 1.26 1.6E−01 6.2E−02 1.0 −1.6 DG 36:2 1.201.34 9.2E−02 2.6E−02 −1.4 −2.0 Coenzyme Q10 0.70 0.92 1.1E−02 3.2E−012.4 0.5 MG 16:0 0.95 1.29 3.0E−01 1.2E−03 0.5 −3.1 MG 18:0 0.88 1.222.8E−02 3.6E−04 2.0 −3.5 Cer d40:1 1.07 0.96 2.4E−01 3.5E−01 −0.7 0.4Cer d42:2 1.63 1.21 5.1E−11 2.3E−02 −7.3 −2.0 Cer d42:1 0.96 0.683.0E−01 3.4E−04 0.5 3.6 PC 30:0 1.48 0.79 1.7E−02 4.1E−02 −2.2 1.8 PCO-32:1/P-32:0 1.89 0.88 5.5E−07 1.5E−01 −5.3 1.0 PC 32:2 0.85 0.601.5E−01 2.1E−05 1.0 4.4 PC 32:1 2.10 0.98 2.0E−06 4.0E−01 −4.8 0.3 PC32:0 1.99 1.12 7.7E−13 5.8E−02 −7.9 −1.6 PC O-34:3/P-34:2 1.02 0.624.1E−01 7.4E−07 −0.2 5.3 PC O-34:2/P-34:1 1.14 0.67 8.5E−02 4.8E−06 −1.44.8 PC 34:3 1.12 0.71 1.0E−01 6.7E−05 −1.3 4.0 PC 34:2 1.28 0.75 1.1E−035.2E−05 −3.2 4.1 PC 34:1 1.88 0.96 1.2E−11 2.9E−01 −7.4 0.6 PCO-36:5/P-36:4 1.04 0.83 3.3E−01 2.2E−02 −0.4 2.1 PC O-36:4/P-36:3 1.140.78 4.2E−02 2.2E−03 −1.8 3.0 PC O-36:3/P-36:2 1.10 0.61 1.9E−01 3.7E−06−0.9 4.9 PC 35:2 1.19 0.75 2.3E−02 1.1E−03 −2.0 3.2 PC 35:1 2.05 1.093.6E−07 2.2E−01 −5.4 −0.8 PC 36:5 0.97 0.73 4.1E−01 8.0E−04 0.2 3.3 PC36:4 1.19 0.86 1.9E−02 4.5E−02 −2.1 1.7 PC 36:3 1.34 0.73 2.2E−049.5E−05 −3.7 4.0 PC 36:2 1.23 0.76 9.0E−03 3.5E−04 −2.4 3.6 PC 36:1 2.101.06 3.6E−06 3.3E−01 −4.7 −0.4 PC O-38:6/P-38:5 1.00 0.82 4.9E−018.0E−02 0.0 1.4 PC O-38:5/P-38:4 1.28 0.93 6.0E−04 2.0E−01 −3.4 0.9 PCO-38:4/P-38:3 1.30 0.88 1.6E−04 6.9E−02 −3.8 1.5 PC 37:3 1.29 0.789.5E−03 2.1E−02 −2.4 2.1 PC 38:7 1.13 0.89 1.3E−01 1.2E−01 −1.2 1.2 PC38:6 1.08 0.91 2.5E−01 2.0E−01 −0.7 0.8 PC 38:5 1.04 0.80 3.6E−017.1E−03 −0.4 2.5 PC 38:4 1.12 0.94 1.1E−01 2.3E−01 −1.3 0.8 PC 38:3 1.250.83 2.4E−02 3.2E−02 −2.0 1.9 PC 38:2 1.68 1.73 1.0E−03 8.4E−04 −3.2−3.2 PC 40:7 1.11 0.97 1.6E−01 3.8E−01 −1.0 0.3 PC 40:6 1.10 1.132.3E−01 1.2E−01 −0.8 −1.2 PC 40:5 1.16 1.05 1.1E−01 2.9E−01 −1.3 −0.6 PC40:4 1.82 1.18 1.0E−03 1.3E−01 −3.2 −1.2 SM d32:1 1.06 0.74 3.1E−018.8E−03 −0.5 2.5 SM d34:2 1.17 0.91 5.0E−03 1.4E−01 −2.7 1.1 SM d34:11.55 0.93 9.9E−10 1.9E−01 −6.5 0.9 SM d36:2 1.30 1.15 2.7E−03 1.0E−01−2.9 −1.3 SM d36:1 1.49 1.16 1.2E−07 5.7E−02 −5.5 −1.6 SM d38:1 0.960.74 2.9E−01 2.0E−03 0.6 3.0 SM d40:2 0.96 0.73 2.7E−01 6.9E−04 0.6 3.4SM d40:1 0.90 0.70 6.7E−02 7.1E−05 1.5 4.1 SM d41:2 0.92 0.69 2.0E−011.1E−03 0.9 3.2 SM d41:1 0.70 0.55 9.2E−05 1.6E−06 4.1 5.1 SM d42:3 1.350.96 5.3E−06 3.2E−01 −4.7 0.5 SM d42:2 1.50 0.98 1.0E−08 3.9E−01 −6.10.3 SM d42:1 0.77 0.61 8.3E−04 2.6E−07 3.4 5.6

TABLE 30 List of all quantified lipids with MALDI-MS measurementtogether with fold changes, p-values, and T-values. Fold change p-valuet-value Lipid M F M F M F cholesterol 1.02 1.21 4.5E−01 1.6E−01 −0.1 −1sulfate cholesterol 1.06 1.69 4.3E−01 6.0E−02 −0.2 −1.6 sulfate (OH) SM32:1 0.86 1.01 1.9E−01 4.7E−01 0.9 −0.1 SM 33:2 1.45 1.31 1.3E−011.7E−01 −1.2 −1 SM 33:1 1.04 1.24 4.2E−01 1.7E−01 −0.2 −1 SM 34:2 1.121.06 2.6E−01 3.6E−01 −0.7 −0.4 SM 34:1 1.19 1.26 1.7E−01 8.0E−02 −1 −1.5SM 34:0 1.13 1.23 2.7E−01 1.5E−01 −0.7 −1.1 SM 35:1 1.15 1.3 2.8E−011.0E−01 −0.6 −1.4 SM 36:2 1.19 1.16 1.8E−01 2.3E−01 −1 −0.8 SM 36:1 1.241.35 1.3E−01 6.0E−02 −1.2 −1.6 SM 36:0 1.42 2 4.8E−02 4.0E−02 −1.8 −1.9SM 37:1 1.08 1.37 3.9E−01 1.6E−01 −0.3 −1 SM 38:1 0.9 1.03 2.9E−014.4E−01 0.6 −0.2 SM 39:1 0.57 0.81 1.7E−02 2.6E−01 2.3 0.7 SM 40:3 1.191.43 2.8E−01 9.5E−02 −0.6 −1.4 SM 40:2 0.94 1.02 3.7E−01 4.7E−01 0.4−0.1 SM 40:1 0.86 0.98 2.1E−01 4.6E−01 0.9 0.1 SM 41:3 1.42 1.26 1.2E−011.9E−01 −1.3 −0.9 SM 41:2 0.84 0.99 2.2E−01 4.8E−01 0.8 0.1 SM 41:1 0.680.85 2.8E−02 2.6E−01 2.1 0.7 SM 42:4 1.16 1.17 2.7E−01 2.7E−01 −0.6 −0.6SM 42:3 1.19 1.19 2.1E−01 1.8E−01 −0.9 −1 SM 42:2 1.19 1.28 2.1E−019.0E−02 −0.9 −1.4 SM 42:1 0.83 0.92 1.7E−01 3.3E−01 1 0.5 SM 43:2 1.271.56 2.5E−01 1.0E−01 −0.7 −1.4 SM 43:1 1.05 0.96 2.5E−01 3.7E−01 −0.70.4 SulfoHexCer 34:2 1.43 0.9 2.5E−01 3.9E−01 −0.7 0.3 SulfoHexCer 34:10.81 0.92 1.8E−01 3.7E−01 1 0.3 SulfoHexCer 34:2 0.86 0.87 3.7E−013.7E−01 0.4 0.4 (OH) SulfoHexCer 35:1 0.63 1.02 2.8E−03 4.7E−01 3.1 −0.1SulfoHexCer 34:1 0.76 0.93 9.0E−02 3.7E−01 1.4 0.3 (OH) SulfoHexCer 35:00.78 1.23 1.9E−01 2.5E−01 0.9 −0.7 SulfoHexCer 34:0 0.58 0.93 4.1E−024.3E−01 1.9 0.2 (OH) SulfoHexCer 37:2 0.96 0.96 3.5E−01 3.3E−01 0.4 0.5SulfoHexCer 37:1 0.62 1.1 4.2E−03 3.6E−01 2.9 −0.4 SulfoHexCer 38:1 1.041.13 4.0E−01 2.3E−01 −0.3 −0.8 SulfoHexCer 38:1 0.94 1.11 3.3E−013.5E−01 0.5 −0.4 (OH) SulfoHexCer 40:2 0.79 0.86 1.6E−01 3.3E−01 1.1 0.5SulfoHexCer 40:1 0.47 0.79 1.0E−02 2.6E−01 2.6 0.7 SulfoHexCer 40:2 0.750.94 1.6E−01 4.4E−01 1.1 0.2 (OH) SulfoHexCer 41:1 0.73 0.76 6.5E−021.9E−01 1.6 0.9 SulfoHexCer 40:1 0.47 0.74 4.8E−03 1.6E−01 2.9 1 (OH)SulfoHexCer 42:3 1.03 1.11 4.7E−01 4.0E−01 −0.1 −0.3 SulfoHexCer 42:20.8 0.96 2.1E−01 4.5E−01 0.9 0.1 SulfoHexCer 41:2 0.8 1.08 1.7E−014.3E−01 1 −0.2 (OH) SulfoHexCer 42:1 0.63 0.74 6.5E−02 2.1E−01 1.6 0.8SulfoHexCer 41:1 0.38 0.74 2.7E−03 2.2E−01 3.2 0.8 (OH) SulfoHexCer 42:31.02 0.98 4.8E−01 4.8E−01 0 0.1 (OH) SulfoHexCer 42:2 0.69 0.89 3.5E−023.0E−01 1.9 0.5 (OH) SulfoHexCer 42:1 0.56 0.76 1.0E−02 1.7E−01 2.5 1(OH) SulfoHexCer 42:1 0.75 1.17 2.0E−01 3.5E−01 0.9 −0.4 (2OH) PI 34:20.9 2.78 4.0E−01 3.2E−02 0.3 −2 PI 34:1 0.82 2.25 2.8E−01 5.0E−02 0.6−1.8 PI 36:4 0.81 2.61 2.4E−01 4.5E−02 0.7 −1.8 PI 36:3 0.89 2.2 2.9E−015.5E−02 0.6 −1.8 PI 36:2 1.02 1.84 4.8E−01 4.9E−02 −0.1 −1.8 PI 36:10.56 1.89 5.0E−02 8.5E−02 1.8 −1.5 PI 38:5 0.79 2.11 4.2E−02 4.4E−02 1.9−1.9 PI 38:4 1.09 1.9 3.7E−01 4.0E−02 −0.4 −1.9 PI 38:3 1.16 1.983.1E−01 6.0E−02 −0.5 −1.7 GM3 34:1 1.35 2.04 1.7E−01 1.2E−04 −1 −4.7 GM340:1 1.07 1.35 3.3E−01 1.4E−01 −0.5 −1.1 GM3 42:2 1.55 2.26 9.5E−027.5E−03 −1.4 −2.7

TABLE 31 Assignment of human male subjects with unknown classificationbased on 4 statistical models (#1 OPLS-DA of shotgun MS, #2 OPLS-DA ofUHPSFC/MS, #3 OPLS-DA of all data, and #4 SVM of all data) and theaverage of all 4 models used for the final assignment (N = normal, T =tumor, T1, T2, T3, T4 = tumor stages (T1, T2 are early tumor stages), Tx= tumor but stage is not reported). Real state = health state/diagnosisconfirmed by standard examination methods (imaging and/or biopsy). FinalAverage Sample predic- Real Model Model Model Model of 4 No. tion state1 2 3 4 models 21 T Tx 1.00 1.00 1.00 0.99 1.00 29 T T4 0.99 1.00 1.001.00 1.00 40 N N 0.19 0.23 0.23 0.08 0.18 44 N N 0.15 0.36 0.18 0.120.20 46 N N 0.26 0.22 0.26 0.13 0.22 50 N N 0.33 0.15 0.27 0.61 0.34 64(T) T4 0.31 0.89 0.40 0.66 0.57 89 T Tx 0.76 0.70 0.80 0.89 0.79 106 TT3 1.00 1.00 0.99 1.00 1.00 110 T T3 0.66 0.44 0.58 0.96 0.66 115 N N0.14 0.48 0.14 0.32 0.27 118 N N 0.37 0.36 0.34 0.09 0.29 120 N N 0.110.42 0.11 0.08 0.18 133 T Tx 0.95 0.73 0.91 1.00 0.90 142 T T3 0.97 0.810.95 1.00 0.93 149 N T2 0.37 0.39 0.38 0.27 0.35 166 N N 0.44 0.30 0.450.43 0.41 168 N N 0.21 0.48 0.23 0.19 0.28 173 N N 0.09 0.46 0.15 0.030.18 177 (N) N 0.54 0.38 0.59 0.28 0.45 184 T T1 0.87 0.93 0.90 0.950.91 194 T Tx 0.82 1.00 0.89 0.99 0.93 203 T T3 1.00 0.83 1.00 1.00 0.96247 T Tx 0.62 0.92 0.61 0.73 0.72 251 T T3 1.00 0.65 1.00 0.99 0.91 255T T4 0.81 0.97 0.89 0.98 0.91 258 T Tx 1.00 1.00 1.00 1.00 1.00 264 T T20.68 0.69 0.60 0.84 0.70 267 T Tx 1.00 1.00 1.00 1.00 1.00 271 T T4 1.001.00 1.00 1.00 1.00 275 T T3 0.98 1.00 1.00 1.00 1.00 305 T T4 0.92 0.961.00 1.00 0.97 310 T Tx 0.83 0.81 0.85 1.00 0.87 317 T Tx 1.00 1.00 1.001.00 1.00 322 T T3 1.00 1.00 1.00 1.00 1.00 347 T T3 1.00 0.71 1.00 0.970.92 351 T T3 0.85 0.74 0.90 0.95 0.86 355 T Tx 0.94 0.73 0.84

TABLE 32 Assignment of human female subjects with unknown classificationbased on 4 statistical models (#1 OPLS-DA of shotgun MS, #2 OPLS-DA ofUHPSFC/MS, #3 OPLS-DA of all data, and #4 SVM of all data) and theaverage of all 4 models used for the final assignment (N = normal, T =tumor, T1, T2, T3, T4 = tumor stages, Tx = tumor but stage is notreported). Real state = health state/diagnosis confirmed by standardexamination methods (imaging and/or biopsy). Final Average Samplepredic- Real Model Model Model of 4 No. tion state Model 2 3 4 models 17T T1 0.89 1.00 0.88 1.00 0.94 25 T T4 1.00 1.00 1.00 1.00 1.00 34 T T31.00 0.98 1.00 0.99 0.99 55 T T4 1.00 0.90 1.00 1.00 0.97 68 T Tx 0.921.00 1.00 1.00 0.98 73 T T3 0.85 1.00 0.89 0.99 0.93 78 T Tx 0.82 0.710.68 0.95 0.79 83 T T3 1.00 1.00 1.00 1.00 1.00 93 T T4 1.00 1.00 1.001.00 1.00 98 T T3 1.00 1.00 1.00 1.00 1.00 104 N Tx 0.16 0.52 0.40 0.230.33 125 T T3 1.00 0.75 1.00 1.00 0.94 130 T T3 0.94 1.00 1.00 0.86 0.95157 T T4 0.61 0.49 0.51 0.96 0.64 161 T Tx 0.92 0.68 0.72 0.88 0.80 181T Tx 0.96 1.00 1.00 0.96 0.98 190 T T3 0.65 0.97 0.67 0.56 0.71 199 N T20.47 0.52 0.44 0.11 0.38 208 T T4 0.76 0.73 0.96 0.97 0.85 218 T T2 0.980.87 0.89 1.00 0.93 226 N N 0.13 0.32 0.22 0.21 0.22 230 N N 0.25 0.360.25 0.02 0.22 235 N N 0.36 0.00 0.31 0.07 0.19 239 (N) N 0.64 0.30 0.480.40 0.46 280 (T) T3 0.33 0.70 0.47 0.56 0.51 283 N N 0.41 0.19 0.300.59 0.37 288 N N 0.16 0.55 0.35 0.22 0.32 292 N N 0.15 0.00 0.00 0.030.04 293 N N 0.00 0.02 0.00 0.01 0.01 296 N N 0.27 0.19 0.20 0.13 0.20327 N N 0.13 0.08 0.11 0.21 0.13 331 T N 0.76 0.33 0.73 0.82 0.66 335 NN 0.00 0.00 0.00 0.00 0.00 344 T Tx 1.00 1.00 1.00 0.99 1.00 359 T Tx0.59 0.64 0.61 0.54 0.59

Calculation of Sample Throughput for Early Screening and Monitoring ofTherapy of Pancreatic Cancer Patients Based on the Lipidomic Analysis ofHuman Serum, Plasma, Urine or Other Body Fluids

Calculation for 100 samples of human serum (plasma, urine, or other typeof body fluid)

Instrumentation:

-   -   1 MS system (shotgun MS, UHPSFC/MS, or MALDI-MS)    -   laboratory for sample preparation in Biological Safety Level        (BSL) 2 regime

TABLE 33 Time calculation of individual steps in our methodology forhigh-throughput lipidomic quantitation Time Step [days] Comments Samplepreparation 1.5 10-12 hours (liquid/liquid extraction) for 1 personAnalysis 2.5 2 injections = 40 samples/ (including continuous 24 hours,1 injection = 80 measurement control) (possible 2. injection incase ofpositive results or ambiguity) Data processing 1 In fact, it is 2.5days, (noise reduction and but it runs in parallel data conversion) withthe analysis Quantitation using 0.75 Microsoft Excel script Multivariatedata 0.25 analysis (MDA) Spare time for 1 unexpected circumstances TOTAL7

Calculation for 1000 samples of human plasma:

7*10=70 working days—reduction due to some multiplexing and possibleover weekend automated operation=ca. 3 months

How many samples per year for 1 MS system:

1000 (per 3 months)*4=ca. 4000 samples/year

Further automation of sample preparation, data processing, multiplexingof tasks, and shortening the analysis time could further increase thesample throughput at least two times.

INDUSTRIAL APPLICABILITY

The method of the invention can be used for population screening of thewhole population or selected population groups based on risk factorssuch as age, gender, body-mass-index, genetic predispositions, riskbehavior, etc. The subjects having the positive output from populationscreening above a pre-determined threshold can then be subjected tofurther examinations and tests (e.g., computer tomography or otheradvanced imaging methods). As the present screening method isnon-invasive, can be performed in a high-throughput mode, and can detectearly stages of cancers, it is the first known method suitable forroutine screening of various cancer types including the early stages.The analytical methodology is fully validated in line withrecommendations of authoritative organizations, such Food and DrugAdministration or European Medicines Agency (EMEA).

1. A method of diagnosing cancer based on lipidomic analysis of a bodyfluid taken from the body of a patient, characterized in that saidmethod comprises the steps of: spiking of the sample with a set ofinternal standards comprising at least one internal standard for eachlipid class present in the sample, subsequently processing the sample byliquid-liquid lipidomic extraction or by solid phase lipidomicextraction, measurement of the processed sample by a mass spectrometrymethod, determining concentrations for at least 51 lipids, morepreferably for all lipids present at a level above detection thresholdof the mass spectrometry method, using the internal standards for thecorresponding lipid classes for the quantitation, or using relativeconcentrations, or using ratios of concentrations and/or signalintensities, statistical evaluation of the determined concentrations ofthe lipids, said statistical evaluation determining the level ofprobability of the patient suffering from cancer, or optionally from aspecific type of cancer, based on the determined lipid concentrationscompared to a cancerous pattern, or optionally a specific cancerouspattern, and a non-cancerous pattern, wherein the cancerous pattern orthe specific cancerous pattern and the non-cancerous pattern aredetermined by statistical analysis of the lipid concentrations for agroup of known cancer samples and non-cancer samples, and wherein thestatistical evaluation is done separately for males and females.
 2. Themethod according to claim 1, wherein the at least 51 lipids include thefollowing lipids: Sphingolipids Cer 34:1 SM 34:2 SM 40:1 SM 42:2 Sul40:1 Sul 42:1 Cer 42:1 SM 36:1 SM 40:2 SM 42:3 Sul 40:1(OH) Sul42:1(2OH) Cer 40:1 SM 36:2 SM 41:1 SM 43:1 Sul 40:2(OH) Sul 42:1(OH) Cer42:2 SM 38:1 SM 41:2 Sul 34:0(OH) Sul 41:1 SM 34:1 SM 39:1 SM 42:1 Sul34:2(OH) Sul 41:1(OH) Glycerophospholipids LPC 16:0 LPC 18:2 PC 34:1 PC36:3 PCO-34:2/P-34:1 PCO-36:5/P-36:4 LPC 18:0 PC 32:0 PC 34:2 PC 36:4PCO-34:3/P-34:2 PE 34:1 LPC 18:1 PC 32:2 PC 36:2 PC 38:4 PCO-36:3/P-36:2Glycerolipids TG 49:1 TG 50:4 TG 50:5 TG 51:4 TG 53:4 TG 55:5


3. The method according to claim 1, wherein pancreatic cancer isdiagnosed based on lipidomic analysis of a body fluid taken from thebody of a patient, characterized in that it comprises the steps of:spiking of the sample with a set of internal standards comprising atleast one internal standard for each lipid class present in the sample,subsequently processing the sample by liquid-liquid lipidomicextraction, measurement of the processed sample by a mass spectrometrymethod, determining concentrations for at least 31 or for at least 51lipids, preferably at least 60 or at least 120 lipids, more preferablyfor all lipids present at a level above detection threshold of the massspectrometry method, using the internal standards for the correspondinglipid classes for the quantitation, or using relative concentrations oflipids, or using ratios of concentrations and/or signal intensities,statistical evaluation of the determined concentrations of the lipids,said statistical evaluation determining the level of probability of thepatient suffering from pancreatic cancer based on the determined lipidconcentrations compared to a pancreatic cancerous pattern and anon-cancerous pattern, wherein the pancreatic cancerous pattern and thenon-cancerous pattern are determined by statistical analysis of thelipid concentrations for a group of known pancreatic cancer samples andnon-cancer samples, and wherein the statistical evaluation is doneseparately for males and females.
 4. The method according to claim 3,wherein the at least 31 lipids include: Lipid HexCer d18:1/16:0; HexCerd18:1/15:1 (1OH) SM 34:1 HexCer d18:1/24:1; HexCer d18:1/23:2 (1OH) PE34:1; PE P-35:0 PC 32:0; PC 0-33:0 PE 36:4; PE P-37:3 PC 34:1; PC P-35:0PE 34:2; PE P-35:1 PC P-34:0; PC 33:1 DG 36:2 Cer d18:1/24:1; Cerd18:1/23:2 (1OH) CE 16:0 SM 41:1 SM 41:2 PC 34:4; PC P-35:3 HexCerd18:1/24:0; HexCer d18:1/23:1 (1OH) PE P-36:3; PE 35:4 PE P-38:4; PE37:5 PE P-38:5; PE 37:6 PE P-36:2; PE 35:3 SM 32:1 HexCer d18:1/22:0;HexCer d18:1/21:1 (1OH) PE P-38:3; PE 37:4 PC P-38:2; PC 37:3 PE P-40:5;PE 39:6 PE 36:0; PE P-38:6 PE 38:0; PE P-40:6, PE 0-39:0 PE P-36:3; PE35:4 PC P-34:1; PC 33:2 PC P-36:2; PC 35:3 DG 34:3


5. The method according to claim 1, wherein the body fluid is selectedfrom serum, plasma, blood, urine, body fluids containing oncosomes,exosomes, extracellular vesicles.
 6. The method according to claim 1,wherein the patient is a mammal, preferably a human.
 7. The methodaccording to claim 1, wherein the internal standards are exogenous lipidcompounds, having the polar head structure typical for the relevantlipid class and containing fatty acyls with shorter chains thannaturally occurring lipids, preferably chains 12:0 or 14:0, or fattyacyls with odd number of carbon atoms, preferably chains 17:0, 17:1 or19:1, or the internal standards are isotopically labelled analogues ofthe lipids of the relevant lipid class, preferably D7-Chol, D7-CE 16:0.8. The method according to claim 1, wherein a set of internal standardscontains: D7-CE 16:0 MG 19:1 Cer d18:1/12:0 PA 14:0/14:0 DG 12:1/12:1 PC14:0/14:0 Hex2Cer d18:1/12:0 PE 14:0/14:0 HexCer d18:1/12:0 PG 14:0/14:0D7-Chol PS 14:0/14:0 LPC 17:0 SM d18:1/12:0 LPE 14:0 SulfoHexCerd18:1/12:0 LPG 14:0 TG 19:1/19:1/19:1 LPA 14:0 LPS 17:1


9. The method according to claim 1, wherein the mass spectrometry methodis selected from shotgun mass spectrometry, ultrahigh-performance liquidchromatography—mass spectrometry, ultrahigh-performance supercriticalfluid chromatography—mass spectrometry, and matrix-assisted laserdesorption/ionization mass spectrometry.
 10. The method according toclaim 1, wherein a pooled sample prepared by mixing identical volumes ofseveral samples is used and processed in the same way as the measuredsamples, wherein the pooled sample includes samples from pancreaticcancer patients and healthy volunteers, and wherein the pooled sample isused for observing intra-day accuracy and intra-day precision, and/orfor inter-day accuracy and inter-day precision, and/or for determiningthe lower limit of quantitation and the upper limit of quantitation,and/or for quality control during measurements of sample set.
 11. Themethod according to claim 1, wherein the order of samples is randomizedin sample measurement sequences.
 12. The method according to claim 1,wherein the step of determining concentrations for all lipids present ata level above lower limit of quantitation of the mass spectrometrymethod, using the internal standards for the corresponding lipid classesfor the quantitation, comprises one or more of the following proceduresand corrections: isotopic correction, zero filling procedure in whichthe signals of lipid species which are not detected for particularsample are replaced by 50 to 100%, preferably by 60 to 100% or by 70 to100%, more preferably by 80 to 90%, or by 80%, of the minimumconcentration observed for said lipid species in all samples.
 13. Themethod according to claim 1, wherein the statistical evaluation involvesdata pre-processing such as centering, scaling, and/or transformation;PCA analysis for identification of influential factors such as outliersor measurement failures, discrimination analysis by OPLS-DA for groupseparation of pancreatic cancer patients and healthy volunteers,preferably performed for males and females separately, assignment of thestatistical parameters as well as the evaluation of the predictionpower.
 14. The method according to claim 1, wherein in a first step ofthe statistical evaluation it is determined whether a tested patientsuffers from cancer based on the cancerous and non-cancerous patterns,and in a second step, provided that the tested patient has a probabilityof suffering from cancer over a pre-determined level, the specific typeof cancer is determined based on the specific cancerous pattern.